Rovers' development squad were once again left to rue an inability to convert their chances as they went down 2-0 to Carlisle United.
Like the match against Fleetwood last month, the young Rovers showed plenty of attacking intent and dominated possession, but they couldn't make it count where it mattered.
Instead it was the resolute and well-drilled Carlisle side who made it back-to-back wins in the Checkatrade Trophy with a goal in each half.
The hosts struck on 39 mintes through Shaun Miller, before Danny Grainger completed the victory from the penalty spot two minutes from time.
In the other group match Oldham's win over Fleetwood means that Rovers can still progress through to the knockout stage, but they'll need to find their goalscoring touch at Ewood in a month's time.
In contrast to the game against Fleetwood where Damien Johnson had named an entire team under the age of 21, the development squad boss was able to call upon some first team experience for the match at Brunton Park.
Wes Brown was handed his first start in Rovers colours after officially joining last month, whilst Elliott Bennett and Anthony Stokes, who have both featured for the Under-23s this term, were also named amongst the starters.
Just three minutes in and Stokes made an early impression. As Reggie Lambe was caught in possession by the Irishman play opened up as he raced forward, but his effort failed to test Max Crocombe in the Carlisle goal.
There was a scare at the other end as Shaun Miller fired goalwards on the turn, but David Raya had to show quick reactions to keep his effort out.
As the clocked ticked on it was Rovers growing in confidence, seeing plenty of ball with their quick incisive passing, and the wing threat of Ryan Nyambe and Connor Mahoney.
But like the match at the Highbury Stadium, Rovers were unable turn their dominance into meaningful opportunities.
After Carlisle had seen a goal chalked off for offside, Rovers did force Crocombe into a save on 38 minutes.
Willem Tomlinson played the ball up to Stokes and he returned the pass to the young midfielder who let fly from 25-yards but the Carlisle stopper leapt high to tip the ball over the crossbar.
However from the resulting corner kick the hosts would break quickly and open the scoring.
A ball out of defence deflected kindly for Carlisle and as Jamie Devitt raced up to the edge of the box he slipped in Shaun Miller who fired past Raya with a composed finish.
That then prompted a purple patch for the home side as they ended the half on the up.
Jason Kennedy was just off target with a shot from outside the box, before Luke Joyce grazed the outside of the post with a speculative effort.
Then only a fantastic one-handed save from Raya prevented Carlisle from doubling their advantage before half-time. Shaun Brisley with the header, the keeper with a marvellous save.
The two sides exchanged chances at the start of the second half. First Stokes showed quick feet to cut in from the left to feed Bennett, but on the stretch the midfielder couldn't get a meaningful connection and Carlisle cleared.
Play was quickly shifted upfield with the lively Jabo Ibehre racing forward, he cut the ball back for Kennedy but his shot was straight at Raya.
Ibehre then fired forward after meeting a cross Danny Granger, but Scott Wharton was in the right place to divert the ball behind.
The resulting corner saw the two Carlisle players combine once more as Granger delivered to the near post, Ibehre nodded goalwards but Raya reacted well to turn the ball aside.
But whilst Rovers continued to play well up to the edge of the box, they again failed to create anything meaningful. Instead the hosts were quick on the counter, carrying the greater threat. Reggie Lambe wasteful in the area with a shot that was high and wide.
But as long as it stayed 1-0 there was always a chance that Rovers could come back into the game.
Stokes headed narrowly over from a Jack Doyle cross, before a cross-shot from Mahoney almost flew into the far corner, but a back-pedalling Crocombe tipped over.
Rovers continued to be camped on the edge of the Carlisle box, Stokes leading the attack time and time again, but their inability to create any meaningful openings would prove costly when the hosts doubled their advantage from the penalty spot with two minutes remaining.
A rare counter attack from Carlisle saw the ball played to the far side of the penalty area and to substitute Cameron Salkeld. As the youngster claimed possession Wes Brown mis-timed his challenge and referee Richard Clark pointed to the spot.
Club captain Danny Grainger stepped up from 12-yards and made no mistake with a shot to the left as Raya dived in the opposite direction.
That knocked the impetus out of Rovers' play as the well-drilled Carlisle defence saw out the remaining minutes to record their second win in the group.
A win for Oldham elsewhere means that Rovers can still progress through to the knockout stage of the competition, but they'll need to defeat Oldham well at Ewood in a month's time and hope that Carlisle claim a victory over Fleetwood in the other fixture.
CARLISLE: Crocombe, Grainger (c), Joyce, Ellis, Kennedy, Ibehre (sub McKee 89 mins), Brisley, Devitt, Lambe, S. Miller (sub Wyke 68 mins (sub Salkeld 79 mins)), T. Miller.
Subs not used: Gillespie (GK), Atkinson, Adams, Brough.
Goals: S. Miller 39 mins, Grainger (pen) 88 mins.
ROVERS: Raya, Nyambe, Brown, Wharton (c), Doyle, Mahoney, Tomlinson, Hardcastle, Bennett, Stokes, Mansell.
Subs not used: Fisher (GK), Travis, Howarth, Platt, Grayson, Rankin-Costello, Magloire.
Attendance: 1,253 (including 49 Rovers fans)