The right-back had to be replaced in the 49th minute of Tuesday night’s 2-1 victory over Scunthorpe which earned a Leasing.com Trophy semi-final spot.
Bolton, who had reported a thigh issue at the interval, was replaced by Christian Burgess at full-back for the remainder of the match.
The summer recruit will today be given a scan to uncover the full nature of the problem.
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And it already raises doubts over whether he will be available for Saturday’s FA Cup visit of Barnsley.
Assistant manager Joe Gallen said: ‘He complained at half-time of a problem with his thigh.
‘We are hopeful it’s not a muscle problem, an indication that it might not be is he didn't quite feel it doing anything dynamic, so it might be a dead leg or something else.
‘If it is a muscle injury, that isn’t good news, but we don’t know, we will find out.’
It’s tough luck for Bolton, who has been given a run at right-back following an uncertain start to his Fratton Park career.
Ross McCrorie, is a potential replacement, yet has still to return from Rangers following treatment of a hamstring injury.
Another alternative could be youngster Haji Mnoga, who lined up for the Blues at right-back at Walsall in the Leasing.com Trophy earlier this month.
Jackett this week admitted he was prepared to let the 17-year-old out on loan to the National League to gain first-team experience.
That could be put on hold, depending on the outcome of Bolton’s scan.