The right-back will today undergo a scan on a hamstring problem collected in Saturday’s 5-1 defeat at West Brom.
He was among five players rested from the starting XI for the trip to The Hawthorns, with Mousinho keen to protect them amid a gruelling fixture schedule.
However, Williams was still introduced from the bench in the 69th minute for Zak Swanson – and subsequently collected a hamstring injury.
The former Barnsley man previously missed eight matches after damaging his hamstring in the second half at Plymouth in November.
Returning to action earlier this month, he subsequently played four times, including three starts, but is now sidelined once more.
Mousinho told The News: ‘Jordan felt his hamstring on Saturday, so we are going to send him for a scan tomorrow and see what the extent of that injury is.
‘It’s a new one, he did it when he came on at West Brom. It’s one of those where we were really trying to protect against injury with the squad rotation and the team rotation.
‘We felt we had to give Connor Ogilvie a rest because he’s been the one we haven’t been able to take at all, so brought Jordan on – but unfortunately he’s picked up an injury.’
Williams has been dogged by injury since his summer arrival from Barnsley on a free transfer.
Previously he has been sidelined by a hip flexor problem and then a previous hamstring issue, restricting him to 17 appearances this season.
Swanson once again line-up at right-back for Tuesday night’s visit of Millwall, with the Blues slipping to a 1-0 defeat through Mihailo Ivanovic’s first-half goal.
That match also marked the welcome return of Conor Shaughnessy, having been absent since August through persistent calf problems.
The popular central defender came on for debutant Hayden Matthews in the 79th minute, receiving a generous reception from the Fratton faithful.
It represented only a third outing of the campaign for the Irishman, whose presence has been much-missed during the battle to remain in the Championship.
Mousinho added: ‘It was really pleasing to see Conor back, it has been six months out, so really happy.
‘Based on the game, Hayden went down with cramp. I thought Hayden played really, really well, so wasn’t necessarily going to take him off, but he started to cramp up and we had Conor on the bench.
‘So it was a great opportunity to bring him on.’
Meanwhile, Rob Atkinson missed the Millwall loss through illness, while Ryley Towler wasn’t included in the squad for ‘selection’ reasons according to Mousinho.
Towler remains under consideration to leave Fratton Park on loan in the current transfer window, following the £1.2m signing of Matthews and Shaughnessy’s long-awaited return for injury.
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