Dembele hurt himself after coming off the bench in the second half of Sunday’s 4-2 win away to Real Sociedad in La Liga.
The injury is different to the hamstring tear that ruled him out for three and half months.
“After the game against Real Sociedad, Dembele had a problem with his left thigh and 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 published on their website.
“The injury will mean he will be out for a period of around three to four weeks.”
He now faces a race against time to be ready for the first leg of Barca’s Champions League tie against Chelsea at Stamford Bridge on February 20.
Dembele only returned at the start of this month after tearing his hamstring in September.
The fitness problems have blighted the start of his career at Barcelona following his 105-million-euro ($124 million) move from Borussia Dortmund in August that could rise to as much as 145 million euros.
Barcelona also said that Andres Iniesta “has a problem in his right calf and his recovery will determine his return to action for the coming matches.”
Iniesta was an unused substitute in Sunday’s game as Barcelona came from two goals down to win 4-2 at Anoeta.
Ernesto Valverde’s side are already unable to call on new record signing Philippe Coutinho until the end of the month after he arrived with a thigh injury following his Spanish record 160 million-euro transfer from Liverpool.