The former Borussia Dortmund winger, who was signed for a fee of €105 million in the summer, missed much of the first half of the season with a similar issue, though Barca have stressed that this problem is with a different part of the muscle.
Dembele, playing in just his fourth league outing for the Catalans, came off the bench to replace Paulinho with 25 minutes remaining, and while he finished the match, succeeded in picking up another issue.
In a statement, the Liga leaders said: "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. The injury will mean he will be out for a period of around three to four weeks."
The 20-year-old has only made seven appearances for the Camp Nou side, totalling 243 minutes. During this time on the field, he has yet to score but has provided a couple of assists.
With January signing Philippe Coutinho, bought for a club record €120m, also on the sidelines for around three weeks, the news comes as a blow to Barca, who lead by nine points over Atletico Madrid after rallying from 2-0 down to beat Real Sociedad 4-2 on Sunday.
Barcelona also confirmed a problem for midfielder Andres Iniesta, but are less certain on his recovery.
"Andres Iniesta, for his part, has a problem in his right calf and his recovery will determine his return to action for the coming matches," they said.