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Rooney, who has scored 34 goals for Man United this season, had only just recovered from an ankle injury when he hurt his groin in training on Thursday.
United beat Tottenham 3-1 without Rooney on Saturday, but the team will have to cope for two more matches without him in its attempt to fight off Chelsea's challenge and win an unprecedented fourth straight league title.
"I think he'll probably take two to three weeks to recover," United manager Alex Ferguson said. "We'll try to get him back. He'll obviously be desperate to get back himself. We'll just have to wait and see."
It is the latest setback for England's most influential player, who missed the two league matches before last week's victory at Manchester City becaue of ankle problems.
The 24-year-old Rooney should be fit a month before England's World Cup opener against United States on June 12.
Another concern for England was the absence of captain Rio Ferdinand on Saturday. The United center back missed a second straight match because of a groin injury after a season plagued by back problems.
England teammate Aaron Lennon returned to action for Tottenham at Old Trafford after four months out due to a groin injury, playing 25 minutes and looking sharp despite the long layoff.
However, the right winger could struggle to prove his fitness to England coach Fabio Capello before the final squad is announced on June 1. Spurs manager Harry Redknapp said Lennon is in doubt for next Saturday's match against Bolton.
