"I felt a pain in my right calf and it got tighter and tighter and I had to leave the pitch," the Brazil international told reporters after the match.
"I think it is the same injury as before but I will undergo some tests tomorrow to see how bad it is."
Ronaldo sustained the original injury when he returned to training after the Christmas break and missed his side's King's Cup match against Athletic Bilbao in midweek, but was deemed fit enough to play against Villarreal.
He has had a difficult season with Real, missing all six of their Champions League matches because of suspension and injury and struggling for fitness after an ankle injury.
"These things happen in football," he said. "If I was a lawyer this sort of think wouldn't occur. What I need now is a change of luck."
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