The 25-year-old centre-back, who had previously missed two months with a similar injury picked up in October, is not expected to return to full fitness until March
Chester limped out of Hull's 1-0 defeat at Norwich City last weekend after just 15 minutes and will now miss at least six weeks of action, manager Steve Bruce has confirmed.
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"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's absence will be offset somewhat by Paul McShane's return to fitness, who missed Hull's last two games with an ankle injury but is in contention for Saturday's FA Cup fourth-round tie at Southend United.
Hull also confirmed the signing of Elliot Kebbie on Thursday.
The former Leeds United youth player, who has previously spent time with Atletico Madrid, will play in the KC Stadium club's development squad after penning a short-term deal.