Messi hurt his left leg during a sprint while challenging for the ball with a Betis defender in the 18th minute.
Barcelona said that Messi had a "muscle injury in his left thigh" and that he would undergo medical tests on Monday.
The four-time world player of the year immediately told his team dugout that he was hurt, and Andres Iniesta replaced him in the 21st. Messi went straight to the bench and sat alongside the rest of his teammates.
"We will see tomorrow," said Barcelona sports director Andoni Zubizarreta. "It's never good news when a player has another injury, and above all when he is named Lionel Messi.
"My opinion is that we can't rush him back."
On Wednesday, Messi had ended a four-game scoring drought— his longest since the 2010-11 season— by netting two goals in a 3-1 win over AC Milan in the Champions League.
Against last-place Betis, Barcelona didn't miss their all-time leading scorer. Cesc Fabregas filled in to set up Neymar's opener before scoring two goals after Pedro Rodriguez had extended the lead.
However, Barcelona may have also lost Fabregas after he left the game in the 82nd with pain in his right knee.
"I started with some pain in my knee," said Fabregas. "I was fine but it got worse as the game went on. I prefer to be cautious because I hurt my knee a few years ago."
Fabregas said that he would also urge Messi to take his time.
"I had the same problem for two years," he said. "You want to come back quickly, but you have to wait. You have to be sure to recover fully from this kind of injury. If not it just keeps coming back."
Messi has missed several games this season with nagging muscle injuries.
He first hurt his left thigh in the Spanish Super Cup in August. He then injured his right thigh in a league match at Almeria in September.
The Argentina star was also sidelined at the end of last season with a right hamstring pull.
While still being his team's top scorer with eight goals in the Spanish league and six in the Champions League, Messi has struggled at times this season to break down opposing defences lacking his usual bursts of speed.