Wanderers came from two goals down to stay clear at the top of their Papa Johns Trophy group but saw Tranmere win the penalty shoot-out to claim the bonus point.
A debut goal from substitute Conor Carty in added time completed the Whites’ second-half fightback as they moved onto four points in Group B.
Carty’s super strike, added to a fine finish of his own from Kieran Sadlier, keeps Wanderers’ destiny in their own hands with a final game to come at home to Leeds United’s U21s in their quest to reach the knock-out stages.
Lloyd Isgrove was the luckless one to fail to convert his penalty as Rovers scored all five of their efforts to win the shoot-out.
But being back on the pitch after 10 months out was bonus enough for Isgrove, who helped the comeback effort as a second-half substitute to help preserve Wanderers’ unbeaten record throughout September.
The Whites had taken maximum League One points during the month and made 10 changes to the side that had made it four victories in a row at the weekend, with only George Johnston asked to go again to extend his ever-present record.
The defender was made captain of a starting line-up which was still full of proven first-team quality, with on-loan Liverpool defender Owen Beck the least experienced as he made his Wanderers’ debut in the left-wing back role.
Josh Sheehan made his second start since returning from a lengthy injury absence as he followed up his return in the opening group win over Crewe.
And the midfielder was close to giving the Whites an early lead with a superbly struck free-kick which almost caught out Tranmere keeper Mateusz Hewelt, who recovered just in time to keep the ball out of the top corner.
But the re-shuffled line-up struggled to gel in a first half which saw Rovers score twice to take control.
Kieron Morris put Tranmere ahead when he strode forward to dive home a skidding effort from 25 yards with 20 minutes gone.
And then after Dapo Afolayan had tested Hewelt again with a rare strike on goal the home side doubled their lead five minutes before the break.
The League Two side had been tidy on the ball and when they worked an opening for Paul Lewis, he made no mistake with a calm finish across Joel Dixon to leave the Whites with it all to do.
The response at the start of the second half was more upbeat as the Whites raised the tempo.
Beck got into a couple of decent crossing positions down the left-hand side and then when Sadlier delivered from the opposite flank, Jon Dadi Bodvarsson flashed a header just wide.
The Whites needed to maintain the momentum but were nearly undone at the other end when Rhys Hughes was given time to measure a shot he steered wide.
Nevitt also missed a chance to make sure of maximum points for Tranmere in their opening group game moments after Evatt made his first change by sending on Isgrove to make a welcome comeback.
Wanderers still needed a spark to trigger a possible comeback and Bodvarsson again almost provided it with a driven effort half stopped by Hewelt and then blocked on the line.
And with 20 minutes to go the Whites’ pressure paid off as Sadlier pulled a goal back.
The chance looked to have gone when Sadlier’s first route to goal was blocked off but when Will Aimson put the ball back into the danger area, Sadlier showed nice control and a fine touch to strike a crash home a volley on the turn from eight yards out.
Time was still against the Whites, though, and with seven minutes of normal time to go Evatt sent on a further striker in Carty to give the former Wolves’ youngster a debut after a recent run of goals in the B team.
It proved an inspired move as the Irishman scored with almost his first touch to equalise in the first of four minutes of added time.
And what a first touch it was as the 20-year-old ran onto a knock-down from the ever-willing Bodvarsson to send a thumping left-foot volley into the back of the net from 20 yards out to set up the penalty shoot-out.
Aaron Morley, Dapo Afolayan, Sadlier and Sheehan all scored.
And although Isgrove saw his effort saved, Wanderers left with plenty of positives to take into the international break.
Wanderers: Dixon; Aimson, Toal (Carty 83), Johnston; John, Morley, Sheehan, Beck (Isgrove 63); Sadlier; Bodvarsson, Afolayan. Substitutes: Hutchinson, Toure, Tweedley, Khumbeni, Stanley.
Booked: None
Tranmere: Hewelt; Dacres-Cogley, Simeu, Byrne, Bristow; Morris (Lomax HT), McAlear, Merrie, Hughes; Nevitt (Burton 68), Lewis (Hemmings 68). Substitutes: Doohan, Turnbull, Hockenhull, Fisher.
Booked: Lewis, Simeu, Lomax
Referee: Martin Coy