The centre-back was hurt in the final minutes of City's 3-2 win at Newcastle on Saturday.
Scans have yet to determine the extent of the problem but manager Steve Bruce is preparing to be without him for a prolonged spell.
"James stretched to make a block in the last minute and his hamstring popped," Bruce told the club's official website.
"It looks like a 'proper' hamstring injury and could be six weeks."
Chester had developed a promising partnership with Curtis Davies this season and signed a new three-year deal at the KC Stadium in June.