The Argentina international was brought off midway through the first half with a mystery problem as his recent fitness issues persist
The Argentine, 26, started in the central role for Gerardo Martino's men in the trip to the bottom-of-the-table Liga side but only lasted 21 minutes - the shortest he has ever featured for the club after being picked in the starting line-up.
Andres Iniesta was brought on in Messi's place, with the four-time Ballon d'Or hobbling off the pitch with no confirmation yet regarding how long the attacker will be out for.
Barca have confirmed that the problem is a hamstring injury to Messi's left leg, with further tests to assess how long he will be out to be undergone on Monday.
He is now a major doubt for Argentina's friendlies in the upcoming international window against Ecuador and Bosnia & Herzegovina.
Messi broke Gerd Muller's record for goals scored in a calendar year in the fixture last season but has now gone five Liga games without a goal.
Barca ultimately won the clash with Betis 4-1 without the forimable forward - Neymar, Pedro and Cesc Fabregas (twice) getting on the scoresheet, with a stoppage-time consolation from Jorge Molina.
Following the match, the Catalans also confirmed that Fabregas was suffering with knee discomfort and would undergo tests soon too.