The central defender was withdrawn after just 15 minutes against Norwich last weekend and scans have revealed he suffered a recurrence of a problem that kept him out earlier in the season.
Chester was sidelined for over two months with the initial complaint and manager Steve Bruce anticipates he will not be ready for first-team action until March.
"James has damaged the tendon again, the one that he damaged at the start of the season," said Bruce.
"It's not as bad as it was the first time he did it, but it's still a concern because it is the same tendon high up in his hamstring.
"He's going to be at least six weeks away I would have thought. It's a shame because he was just finding his feet again after his last spell out, but that is football."
Chester, who turned 25 on Thursday, has been a first choice when fit this season and scored his only goal of the campaign against former club Manchester United on Boxing Day.
Abdoulaye Faye is on hand as cover, though his lack of pace is a concern against some sides, meaning Paul McShane's return from an ankle injury comes at a welcome time.
"Paul is back in full training and knocking on the door," added Bruce.
"It's been a stop-start season for him unfortunately with being in and out of the team having picked up a couple of injuries. Let's hope that he can stay injury-free from now until the end of the season because he can add something to us defensively."
Bruce may yet be tempted to look to recruit in the January transfer window, though the B£14million splurge on Nikica Jelavic and Shane Long accounted for most of his budget.
One new arrival was confirmed on Thursday, with Halifax born right-back Elliot Kebbie signing until the end of the season.
Kebbie, 19, has been training with the club for a month since his release by Atletico Madrid, where he failed to make the grade having signed as a youngster.
He is unlikely to feature in the first team picture this term and will instead bid for a longer deal in Stephen Clemence's development squad.
Clemence said: "Elliot initially came in to train with us to keep his fitness up and we ended up giving him a trial.
"He's an athletic player with speed and a player who is looking to improve. He's got a contract until the end of the season and we'll assess him over the next few months.
"Elliot has certainly got the right attitude and we wouldn't bring anyone in who we didn't think had the right work ethic. This is a big few months for Elliot, but we're hoping he can do well."
Meanwhile, out-of-favour striker Nick Proschwitz has joined Barnsley on loan until the end of the season.