Grimsby Town's Checkatrade Trophy campaign will not go beyond the group stage after they lost an entertaining local derby to Scunthorpe United at Glanford Park.
An almost entirely changed Mariners side played some good football during the first half and took a deserved lead through Diallang Jaiyesimi, only to be pegged back when Tom Hopper equalised for the hosts just before half-time.
After the break, Scunthorpe stepped up a gear, although it took until 10 minutes from time for the League One side to go ahead, with Hopper's second goal of the game eventually proving to be the winner.
And after Town picked up only one point from their opening group game against Doncaster Rovers, the South Yorkshire side's victory over Sunderland Under-21s means they will join Scunthorpe in the first knockout stage of the competition, with one group fixture still to play.
Ten changes were made to the Town side that drew with Lincoln City on Saturday, with only Danny Collins remaining in the starting XI from that game.
Ben Killip started in goal, with Karleigh Osborne, Zak Mills and Collins forming a back three as Tom Bolarinwa and Sam Kelly operated as wing-backs.
Harry Clifton, Mitch Rose and Jaiyesimi were the midfielders, with Harry Cardwell starting alongside JJ Hooper in attack.
The substitutes were James McKeown, Paul Dixon, Sam Jones, Scott Vernon, Martyn Woolford, Siriki Dembele and Jamille Matt.
Scunthorpe boss Graham Alexander made seven changes after their defeat at League One leaders Shrewsbury Town on Saturday but still named a strong line-up featuring the likes of Paddy Madden, Hopper and Kevin Van Veen, who would all look to cause a serious threat to the Grimsby defence.
An early cross from Bolarinwa on the right was cut out, before a low ball from the same side was comfortably gathered by home keeper Rory Watson.
A Scunthorpe corner from the left was dealt with, then Hooper looked to turn in the box at the other end but overran the ball and a goal kick was the result.
Town won a free kick on the left on nine minutes but they were unable to turn it into an opportunity in front of goal, with the offside flag going up as Kelly's ball came across.
Sam Mantom let fly with a shot fom 25 yards soon afterwards which was straight at Killip, while the keeper had to come off his line soon afterwards to deny Hopper.
Another Grimsby free kick, this time from the right, led to Cardwell's header being deflected over the bar for a corner and a shot from Clifton was blocked from it before Jaiyesimi's follow-up flew high and wide.
Town's best chance of the game so far came on 20 minutes when Bolarinwa set off on a run at pace through the middle. He found Hooper but the striker's effort from the right of the penalty area went just over the bar.
A header from Jaiyesimi was caught by Watson soon afterwards, before Kelly beat right-back Levi Sutton down the Town left but his low ball into the box was cleared.
A Bolarinwa crross from the right just evaded the head of Cardwell on 25 minutes as the Mariners continued to do most of the attacking.
But Scunthorpe had an opportunity of their own soon afterwards when a shot from he edge of the box by Madden was blocked for a corner and from the flag-kick, Van Veen planted a header wide when unmarked on the edge of the box.
Hooper headed tamely wide at the other end as the half-hour approached, before the Mariners took a deserved lead on 32 minutes. Kelly and Hooper linked up before JAIYESIMI got the ball inside the box and worked an opening then slotted past Watson.
The hosts looked for an almost instant reply but Hakeeb Adelakun put his shot wide from 20 yards, then a cross from the Scunthorpe left was headed clear by Collins.
More good play by the Mariners came four minutes before the break when a clever ball from Jaiyesimi found Cardwell inside the box, although the striker's shot from a narrow angle was blocked.
But when the second goal of the game arrived on 43 minutes, it was the home fans who were cheering a Scunthorpe equaliser. A shot from Van Veen was blocked and after Town failed to fully clear their lines, HOPPER shot through a crowd and into the net from 20 yards to make it 1-1.
One added minute was signalled to be played at the end of the half and Town aimed to grab the advantage back before the interval, with a shot from Rose being deflected behind for a corner.
The flag-kick was played short in what proved to be the last action of the half, meaning the teams went into the dressing rooms with the scores level.
No changes in personnel were made before the restart, although Town appeared to revert to a 4-4-2 formation with Mills moving from the centre of defence to his more familiar right-back slot as Osborne and Collins took up the central roles.
An early Grimsby free kick from Mills came to nothing but the pressure continued with a corner on the right soon afterwards, which saw Watson save Hooper's far-post header.
A foul on Clifton earned mifielder Devonte Redmond the first yellow card of the game but th resulting free kick went straight through to Watson.
A chance for Scunthope then came when Van Veen found Adelakun but his shot from inside the box was stopped by Killip.
Madden also succeeded only in finding Killip with an effort from inside the box on 53 minutes, before a Kelly cross was headed over the bar by Clifton at the other end.
The home side went close again when a cross from Lewis Butroid led to Madden's header being tipped over the bar for a corner by Killip. The striker was involved again when Town failed to clear the ball from the flag, although his overhead kick went well over the bar.
Madden saw another effort deflected wide for a corner a minute before the hour, with Osborne heading the delivery from the flag clear.
Mills battled well to win a corner at the other end two minutes later but Hooper could only head wide from Bolarinwa's powerful delivery.
Soon afterwards, a foul on Rose brought a free kick inside the Scunthorpe half but Mills' delivery was also headed out for a goal kick.
A powerful run from Bolarinwa down the right brought another Town corner on 67 minutes but the winger's ball from it was cleared.
Mantom fired just wide from the edge of the box for Scunthorpe, before another attack frrom the hosts was thwarted when Madden's volley was saved by Killip.
Woolford replacd Mills in the first substitution of the game with 20 minutes to go, with Clifton moving to right-back.
A cross-shot by Hopper was deflected away from the path of Madden and out for a corner as Scunthorpe continued to threaten, then Van Veen shot wide of the far post from the home side's next chance.
Dembele replaced Jaiyesimi with 13 minutes left on the clock, before Scunthorpe's recent pressure was finally made to tell when they took the lead for the first time in the game on 80 minutes. A corner from the left was not dealt with by the Mariners and HOPPER was the man to eventually head the ball past Killip.
Vernon came on for Hooper as Russell Slade made his final change with eight minutes left, but it was Scunthorpe who were now doing most of the attacking as they looked to seal their victory with a third goal.
Four added minutes were signalled as Van Veen was booked for a foul on Cliifton, before a shot from Dembele was blocked as Town looked for a late leveller.
But it was not to come as Scunthorpe saw out the remainder of the game to ensure their place in the next round of the competition with a game still to play.
The Mariners are next in action on Saturday, when they travel to Port Vale in the league.
31 Rory Watson
22 Levi Sutton
33 Lewis Butroid
21 Cameron Burgess
5 Murray Wallace (C)
8 Sam Mantom
16 Hakeeb Adelakun
18 Devonte Redmond
10 Kevin van Veen
14 Tom Hopper
9 Paddy Madden
1 Matthew Gilks
23 Rory McArdle
12 Neal Bishop
19 Duane Holmes
44 George Hornshaw
11 Josh Morris
17 Lee Novak
30 Ben Killip
6 Danny Collins (C)
14 Karleigh Osborne
12 Zak Mills 71'
18 Tom Bolarinwa
11 Sam Kelly
8 Mitch Rose
15 Harry Clifton
20 Diallang Jaiyesimi 78'
9 JJ Hooper 82'
17 Harry Cardwell
1 James McKeown
3 Paul Dixon
27 Siriki Dembele 78'
10 Sam Jones
25 Martyn Woolford 71'
29 Jamille Matt
21 Scott Vernon 82'
Attendance: 1987 (167)
Referee: Tom Nield