Lionel Messi will be out from between six and eight weeks after being withdrawn due to injury for the third time this season in Barcelona's clash with Real Betis, which the Catalans won 4-1 to extend their advantage in La Liga.
Messi pulled up as he tried to accelerate with the ball at his feet before being replaced by Andres Iniesta.
His club confirmed on Monday that the 26-year-old will miss the final two matches in the Champions League group stage for Barcelona, against Ajax Amsterdam and Celtic.
Barca have already qualified for the last 16, which takes place in late February.
Messi will also miss Argentina's friendlies against Ecuador, on November 15, and Bosnia, on November 18.
The four-time World Player of the Year has suffered from a series of muscular problems since pulling a hamstring against Paris Saint-Germain back in April.
He returned from that injury just 10 days later to help Barca past the French champions and into the semi-finals of the Champions League.
However, his season was ended by a recurrence of the injury against Atletico Madrid in May.
The Argentine was also substituted with a hamstring problem against Atletico in August and pulled up with a thigh problem in Barca's win over Almeria at the end of September.
Messi has failed to score in La Liga since returning from that injury, marking his longest run without a league goal in over two years.
However, he had appeared to have returned to form when he scored twice in Barca's 3-1 win over AC Milan in midweek.
In his absence on Sunday, Neymar, Pedro and two goals from Cesc Fabregas gave Barcelona a 4-1 win over Betis that put them three clear of Atletico Madrid.
Atletico had been denied a couple of hours on top of La Liga when Juanfran put the ball in his own net late on in a 1-1 draw at Villarreal earlier.
Diego Simeone's side were on course for a victory after a Villarreal own-goal in the second minute.
Juanfran's cross forced Mario to head into his own net under pressure from Koke, and the visitors then put in a typically abrasive performance in an intense tussle.
Villarreal were finally rewarded for their endeavour when Jeremy Perbet got to the line and crossed to the near post where striker Ike Uche slid in with Juanfran, and the Atletico defender poked the ball over the line in the 79th.
"We were facing very strong opponents that are playing very well and are in good form," Simeone said.
"Defensively we did very well. We didn't have clear chances, but we did control the ball.
"In the second half we improved a bit but it is an important point against a rival for the Champions League positions. I congratulate the players for the effort they are putting in."
Second-placed Atletico moved on to 34 points from 13 games.
Real Madrid sit third with 31 points after Cristiano Ronaldo scored a hat-trick in their 5-1 home rout of Real Sociedad on Saturday. Promoted Villarreal are fourth with 24.
Earlier, there were whistles at Mestalla after Miroslav Djukic's Valencia came from behind to draw 2-2 against his former club Real Valladolid with a 75th-minute strike from substitute Sofiane Feghouli.
The hosts got off to a woeful start, Ever Banega hoofing a penalty over the bar after only five minutes, and they then conceded a ninth-minute Javi Guerra header.
Colombia striker Dorlan Pabon's long-range effort put Valencia level in the 29th, and after Valladolid notched a second through Gilberto Garcia just after the re-start, Feghouli rescued a point for Djukic's inconsistent side.
Sevilla ended a run of 22 consecutive road games in La Liga without a win when they triumphed 3-1 at Espanyol.
The Andalusians last won an away league fixture in September 2012, beating Deportivo La Coruna 2-0, and coach Unai Emery was feeling the heat after five games without a win in all competitions.
Quick goals from Federico Fazio and Vitolo gave Sevilla the advantage and Carlos Bacca made sure of the points with an impressive third in the 58th minute, running from the halfway line to fire home.