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.
13. Kyle Hurst - 7
Provided the cross for Miller's consolation goal. Like Miller, helped provide some much-needed attacking threat following his introduction. Photo: Howard Roe/AHPIX LTD
14. George Miller - 8
Rovers looked sharper in attack following his half-time introduction and the striker grabbed his seventh and eighth goals of the season with two smart finishes. Photo: David Munro/AHPIX LTD
15. James Maxwell - N/A
Introduced with the clock ticking but made no real contribution. Photo: HOWARD ROE, Howard Roe/AHPIX LTD
16. Kyle Knoyle - 6
Introduced in a double switch and quickly looked to get Rovers up the pitch. Photo: Ben Roberts/AHPIX LTD