Villa netted twice in Thursday's 5-0 Cup win against second-division Cordoba at the Nou Camp, which sent Barca through to the quarter-finals 7-0 on aggregate, but was taken off near the end as a precaution after pulling up and clutching the back of his right leg.
"David Villa will miss the last game of the first half of the season," Barca said.
"The Asturian forward noticed some discomfort during this afternoon's training session," the club said, adding that it was unclear how long he would be out of action.
After returning from a broken leg which sidelined him for the second half of last season, Villa has started only six of the 13 league games in which he has featured and only one game in the Champions League.
The 31-year-old Spain international, the world and European champions' record scorer, competes for a slot up front alongside Lionel Messi with the likes of Pedro and Alexis Sanchez.
There was better news for Barca coach Tito Vilanova about full back Adriano, who was declared fit on Saturday.
Xavi, Andres Iniesta, Gerard Pique and Pedro also return to the squad after they were rested for the Cup game.
With 18 of 38 matches played, unbeaten Barca are 11 points clear of second-placed Atletico Madrid, who host Real Zaragoza on Sunday.