The French winger, signed last year from Borussia Dortmund for a fee which could rise to 147 million euros, suffered a hamstring tear in September that kept him out until January.
Dembele featured in four games after returning, starting one, before suffering a new problem in the 4-2 win over Real Sociedad on Sunday.
"Tests have confirmed that the player has suffered a hamstring injury of the semitendinosus muscle that does not affect the area operated on earlier in the season," Barcelona said in a statement.
Dembele will miss King's Cup and La Liga matches but should return before the Champions League last-16 clash against Chelsea at Stamford Bridge on Feb. 20.
Captain Andres Iniesta, who was on the bench against Real Sociedad, also has a problem in his right calf and is a doubt for the King’s Cup match at Espanyol on Wednesday.
(Reporting by Rik Sharma, editing by Ed Osmond)
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