Madrid: Lionel Messi has sustained a strain in his left thigh, Barcelona confirmed on Wednesday.
Messi was substituted at half-time of Barca's 2-1 win over Villarreal on Tuesday in what was the striker's first start of the season.
He had previously been out with a calf injury sustained on 5 August and Barcelona have not indicated when they expect their captain to return from his latest setback.
"First team player Leo Messi has a strain in the adductor of his left thigh," a club statement read. "He is out and his recovery will dictate his availability."
Messi is expected to miss his team's visit to Getafe in LaLiga on Saturday.
Barcelona then play at home to Inter Milan in the Champions League next Wednesday before hosting Sevilla in the league four days later.
They will hope to have the 32-year-old back as soon as possible, particularly after a rocky opening to the season that has included only three wins from seven matches in all competitions.
After recovering from his calf problem, Messi made substitute appearances against Borussia Dortmund and Granada before being deemed ready to start against Villarreal.
He set up the opening goal, providing the cross for Antoine Griezmann to head in after six minutes. Despite Messi going off at the interval, Barca secured a comfortable 2-1 victory at Camp Nou.