A Joe Wright own goal gave Town the lead at Doncaster but Cameron John and skipper Ben Whiteman have given the home side a 2-1 half-time lead over the Blues.
Town boss Paul Lambert made one change from the team which beat Accrington 2-0 on Saturday with Jon Nolan replacing Teddy Bishop, who dropped to the bench, in midfield.
"Striker Oli Hawkins, who missed the weekend match due to a groin problem, was also among the subs.
Lambert started his front three as they moved to in the second half on Saturday with Freddie Sears down the middle, Gwion Edwards on the left and Alan Judge on the right.
Doncaster made two changes from the team which beat Portsmouth 1-0 at Fratton Park on Saturday with veteran former Town target James Coppinger and Reece James moving to the bench, while Matt Smith and Josh Sims made their full debuts having been subs against Pompey.
After the players took a knee in support of Black Lives Matter, the Blues dominated possession in the opening couple of minutes.
Sears won a seventh minute corner for Town following good work from Edwards and Emyr Huws on the counter-attack and from the flag-kick the ball briefly ran loose but to a Rovers player and the home side were able to break.
On nine Huws brought the ball forward midway inside the Doncaster half but scuffed his shot through to Doncaster keeper Josef Bursik.
Neither side had been able to create a more serious chance than that in an evenly-balanced opening spell until the Blues went in front right on the quarter-hour mark.
Skipper Chambers won a header on the right from a Tomas Holy long kick, then burst forward on to Judge’s return pass before sending in a low cross which Joe Wright turned into his own net ahead of Sears.
There was a big scare for the Blues in the 21st minute when Holy scuffed a pass forward to Sims not far outside the area. Nsiala got across quickly to make an important challenge, then Smith curled a shot over.
But Town were seeing most of the ball and passing it around confidently. In the 26th minute Sears and Judge combined on the edge of the box and the Irish international shot into Bursik’s arms.
However, in the 28th minute Rovers levelled with their first shot of the game following a sustained spell of passing in the Town half.
Eventually, with Nsiala and James Wilson having gone after the same ball outside the box, Smith played Cameron John in in space on the right of the area and the former Wolves trainee found the net off the underside of the bar.
Having conceded for only the third time in the league this season, the Blues went about restoring their lead. On 33 a Judge cross from the right was headed straight up in the air by a Doncaster defender ahead of Bursik but the ball failed to fall kindly for Town.
Two minutes later at the other end, Madger Gomes hit a low shot which flew not too far wide.
And in the 37th minute the home side went in front in a somewhat freakish manner. A short corner on the left was played to Sims and on to Whiteman, who sent a ball into the box from midway inside the half which evaded everyone and bounced past Holy to his left and into the net.
Town were behind for the first time in the league this season having not previously conceded twice in a game. Rovers continued to push after their second goal, a dangerous ball from the left flying across the area.
In the 40th minute, Judge hit the wall with a free-kick from not far outside the area and Chambers was unable to make anything of the loose ball.
Two minutes later, Judge found Huws on the edge of the area from where the Welshman hit a powerful volley over the bar.
Town made their way down the tunnel behind at the break for the first time in the league this season.
Having started well enough and got their noses in front, the Blues conceded two disappointing goals against a Rovers side which had previously looked unthreatening.
Both centre-halves were pulled into the same area for the first after Doncaster had been allowed to keep hold of the ball for a long spell, while the second should never have found its way into the net.
From there, Rovers looked the more confident side with Town unconvincing in the latter stages.
Doncaster: Bursik, Halliday, Anderson, Wright, Gomes, Whiteman (c), Okenabirhie, Taylor, Smith, Sims, John. Subs: Jones, James, Lokilo, Richards, Williams, Butler, Coppinger.
Town: Holy, Chambers (c), Nsiala, Wilson, Kenlock, Dozzell, Huws, Nolan, Judge, Edwards, Sears. Subs: Cornell, McGuinness, Woolfenden, Lankester, Bishop, Bennetts, Hawkins. Referee: Scott Oldham (Poulton-le-Fylde).