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.
9. Harrison Biggins - 5
Hit the bar in the second half but so often goes missing for large spells. Photo: HOWARD ROE, Howard Roe/AHPIX LTD
10. Aidan Barlow - 4
Offered nothing in attack in a toothless first-half display. Not the only one to go missing, in truth, but can have no complaints being down the pecking order. Hooked at the break. Photo: HOWARD ROE, Howard Roe/AHPIX LTD
11. Reo Griffiths - 5
Never looked particularly threatening up front before being replaced at the break. Photo: HOWARD ROE, Howard Roe/AHPIX LTD
12. Jon Taylor - 4
Huffed and puffed but was largely ineffective and barely troubled Barnsley's defence. Photo: HOWARD ROE