The Brazilian forward has only just recovered from the cracked vertebra he suffered at the World Cup finals and was back in full training ahead of Sunday's Primera Division match at the Nou Camp.
But a statement published on Barca's official website read: "Following the Thursday training session Neymar complained about some pain in his left ankle, and tests have confirmed that the player has a Grade 1 ankle sprain.
"The club's medical team has indicated that his return to training will depend on how the injury treatment evolves."
The fresh setback comes only days after the 22-year-old scored twice in a 6-0 friendly victory over Leon.
The statement continued: "It's worth recalling that, last Monday, Neymar received his medical discharge from the back injury that he suffered during the World Cup in Brazil, and was able to take part in the Joan Gamper Trophy match on the same day."
Barca were unable to offer a prognosis on Friday morning but it is likely Neymar will struggle to recuperate in time to face Villarreal at El Madrigal on August 31.