The 25-year-old left-back, who was returning to the team after a two-game absence, picked up the problem in the first half at Elland Road on Saturday and had to be withdrawn at the interval.
The Millers are likely to have had the injury scanned, but are yet to reveal the extent of it.
"Joe Mattock has a hamstring strain," boss Neil Redfearn said after the game on Saturday. We don't know the degree of
it.
"It's disappointing because I thought he played well at Leeds."
Mattock's injury rap sheet is quickly lengthening as this is the fourth problem picked up since joining in the summer.
He suffered a shoulder injury in pre-season which saw him sidelined for the start of the season before missing a month of action after picking up a foot problem in just his second game. He then limped out of Redfearn's first game in charge with another shoulder injury.
And this latest problem looks set to keep him out of the side for at least a couple of weeks, though the extension of Harry Toffolo's loan ensures the Millers are well covered in that area.
Another area on the pitch where there is plenty of options is centre-back after the new pairing of Kirk Broadfoot and Stephen Kelly excelled at Elland Road.
Kelly, making his debut, and Broadfoot, starting for just the second time this season, marshalled the defence superbly and ensured the Millers registered just their third clean sheet of the campaign.
And with Kelly able to occupy a central role as well as either full-back position, Redfearn is enthused.
"I thought he was outstanding," Redfearn said of Kelly.
“The thing is with Kells, he can play anywhere across the back four.
"What he does is give you experience, and he’s quick so he gives you pace too. I thought Broadfoot was outstanding as well."
