Real did not say how long the Portugal captain, who has also been suffering from a knee problem, would be sidelined but local media reported he would almost certainly not be available for the final at Valencia's Mestalla stadium, a repeat of the 2011 showpiece when Ronaldo scored in Real's 1-0 win.
"Medical tests performed on Cristiano Ronaldo have revealed an injury to the player's left biceps femoris muscle," Real said in a brief statement on their website.
Ronaldo was rested for Real's Champions League game at Borussia Dortmund on Tuesday, when the Spanish club lost 2-0 but went through to the semi-finals 3-2 on aggregate.
Spanish media reported he had picked up the thigh strain in a training session the previous day, with sports daily As saying he would be out for two weeks.
The two-legged Champions League semi-finals are on April 22/23 and April 29/30. Real are third in La Liga, three points behind leaders Atletico Madrid, and host Almeria on Saturday.
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