Rovers’ hopes of progression out of the group in the Papa John’s Trophy were dealt a severe blow as visitors Barnsley surged out of sight in the first half on their way to a 4-2 triumph.
The visitors ruthlessly ran riot in the opening period, with former Rovers loanee Josh Martin and youngster Fabio Jalo both scoring twice.
Rovers responded well after the break with substitute George Miller returning from injury to grab a consolation brace.
Gary McSheffrey made eight changes to the side that started the draw with Leyton Orient in league action four days earlier, with Tom Anderson making a welcome return to action as he continued his recovery from injury.
After a few minutes of sizing each other up, both sides began to move the ball quickly, resulting in an open start to the tie.
The visitors took the lead 11 minutes in when Danny Benson pounced on a miscue at the back and clipped the ball across the box where Martin slotted home from seven yards.
Rovers produced some crisp build-up play and Griffiths was inches away from grabbing the equaliser as a Luke Molyneux ball across goal flashed past him.
Slobodan Tedic dropped a potshot narrowly over the bar after meeting a direct ball from the visitors.
The lead was doubled on 20 minutes as Martin capped off a swift build-up by feeding the ball into 16-year-old Jalo, who spun and drilled a low effort into the bottom corner.
And six minutes later, Barnsley extended their lead. More crisp passing play from the Reds allowed Martin to find Jalo on the left of the box, with the youngster able to shift the ball onto his right foot and find the far corner.
Rovers struggled for any meaningful possession as they attempted to find a way back into the game.
And hopes were further dashed on 34 minutes. Ro-Shaun Williams scuffed an attempted clearance and Martin pounced, set himself 15 yards out and fired into the bottom corner.
Rovers’ night looked to have got even worse on 39 minutes when Tedic broke clear from half way and, in his attempts to stop the towering striker, Anderson clipped him inside the box with referee Marc Edwards awarding a penalty.
But Louis Jones got down superbly to push away the subsequent spot kick from Tedic.
And Jones denied Tedic again a minute later as he blocked a close range header low on his line.
McSheffrey sent on George Miller and Kyle Hurst at the break in a bid to inspire a comeback.
The first opportunity was crafted two minutes into the second period when Taylor latched onto a ball into the box only to see his low shot blocked.
Rovers began to enjoy more possession in opposition territory and quickly had the lion’s share of possession.
Miller got away from the Barnsley defence and drilled a shot which Jack Walton pushed over the bar. And moments later the Reds keeper almost dropped into his own net after being put under pressure by Miller, but the ball was hacked clear off the line.
The striker found joy on 73 minutes when he rose to flick a header from a Hurst cross and loop the ball over Walton into the far corner.
Jones had to be alert to push away an effort flicked on goal from a corner, while Jalo fired over the bar from close range with his follow-up.
Thoughts of an unlikely comeback were stirred on 81 minutes when Miller grabbed a second. He surged forward on the left, exchanged passes with Molyneux and drilled home from the edge of the box.
Barnsley were close to a fifth when Luca Connell smashed a shot from 25 yards which deflected narrowly wide off Williams before the game somewhat petered out in the later stages.
Rovers must now await the result of the final tie in the group between Lincoln City and Newcastle United U21 on October 18. Newcastle must win, or take the bonus point in the shootout in the event of a draw, for Rovers to progress to the knockout stages.
Rovers: Jones, Williams, Anderson (Knoyle 63), Long, Seaman, Ravenhill, Biggins, Molyneux, Taylor (Maxwell 63), Griffiths (Miller 46), Barlow (Hurst 46). Subs not used: Mitchell, Faulkner, Close.
Barnsley: Walton, Edwards (Pickard 60), Lancaster, Moon, Larkeche (Hartley 78), Benson, Phillips (Nejman 60), Connell, Martin, Tedic (Dyer 78), Jalo (Butterfill 90). Subs not used: Searle.