Rovers, who were top of Group E at kick-off, conceded four first-half goals and gave away a penalty as they were embarrassed by what was believed to be Barnsley's youngest-ever side in the opening period.
Sixteen-year-old Fabio Jalo helped himself to two goals for the visitors along with former Doncaster loanee Josh Martin.
A second-half brace from substitute George Miller helped salvage some pride, however, with the game finishing 4-2 to Barnsley, who secured qualification.
Doncaster now need Newcastle’s under-21s to take points from Lincoln to qualify.
5. Ro-Shaun Williams - 5
Stepped out of defence with the ball once or twice in the first half as he tried to make something happen but fluffed a clearance for the fourth goal. Made some late interceptions to keep Barnsley at bay in an improved second-half display. Photo: Ben Roberts/AHPIX LTD
6. Charlie Seaman - 4
Two first-half goals came from the right-hand side where he was defending from. Got forward regularly in the second half as Rovers salvaged some pride. Photo: Howard Roe/AHPIX LTD
7. Luke Molyneux - 4
Can't remember him producing anything of note all game. Photo: Ben Roberts/AHPIX LTD
