Goals either side of the half from Grimsby Town resulted in defeat for Rovers' U18s at Cantley Park.
Manager Gary McSheffrey was forced into a change before the game had started, with Michael Nesbitt pulling out of the warm-up and was replaced in the starting XI by Charlie Petch.
The disruption to McSheffrey’s team continued with Owen Scattergood having to come off for Corie Cole 15 minutes into the game after the attacker was on the receiving end of a tackle.
For the majority of the first-half Rovers had some good spells of possessions on the ball, but lacked a bit of cohesion.
When Grimsby did hit Rovers on the break, their captain Harvey Tomlinson was stopped in his tracks by a perfectly-timed tackle by Bobby Faulkner.
Jack Goodman had a couple of chances as he advanced into a dangerous position he was wrestled off the ball by George Sims before he had a chance to pull the trigger. His second chance came from a brilliantly worked counter-attack which finished with the striker's shot being blocked.
Grimsby took the lead minutes before half-time after Taz Goundry broke free of his man to tap in past Tom Chambers from Tomlinson’s free-kick.
A change of system at the break led to McSheffrey withdrawing defender Petch for attacker Faris Khan.
Minutes after the start of the second period, Rovers were 2-0 down after Faulkner misplaced the ball and gave it to Edwin Essel who galloped around Chambers to double the visitor's advantage.
Grimsby worked hard on and off the ball from this point to persevere their lead and protect their clean sheet, with only a couple of chances coming Rovers’ way. Goodman gave his side some hope by forcing their goalkeeper into a save after shrugging off his man.
The final chance of the game for Rovers saw substitute Corie Cole’s shot blocked by Goundry on the 18-yard line.
Rovers return to action at home to Bolton Wanderers on August 22 in the first game of the EFL Youth Cup.