Rovers Under-18s couldn't back up last weekend's victory over Burnley after going down at home to Middlesbrough at the Academy on Saturday.
Brandon Lonsdale scored late on, but it wasn't enough as the visitors, flying high towards the top of the table, ran out comfortable winners.
Boro raced into a 2-0 lead before the break, with Paul Steven opening the scoring for shortly before the half-hour.
Soon after and and George Waites doubled the advantage to put Mark Tinkler's side in command at the interval.
Rovers needed a response, and it came late on when Lonsdale halved the deficit with only two minutes of the contest remaining to give the hosts hope.
Pressing for what would have been a late equaliser, Rovers wer stung at the other end when Calum Kavanagh latched onto John Fenton's pass to beat Jordan Eastham and restore the two-goal cushion just seconds before the final whistle.
Attentions now turn to the cup for Mike Sheron's youngsters, with an FA Youth Cup semi-final tie that was initially scheduled at the end of last season, against Manchester City Under-18s on Friday 30th October.
Kick-off is at 5pm at St George's Park in Burton-upon-Trent, with the game being played behind closed doors.
Rovers Under-18s: Eastham, Haddow, Pratt (Wood, 61), Connolly (Wyatt, 48), Garrett, Pleavin, Weston (Wharton, 75), Harlock, Baker, Gilsenan, Lonsdale.
Unused substitutes: Blease, Gamble.
Middlesbrough Under-18s: Metcalfe, Wilson, Collins (Doherty, 73), Reed, Steven (Fenton, 80), Hannah (Lindo, 59), Waites, Coburn, Willis, Stott, Kavanagh.
Unused substitites: Swann, Hutchinson.