The FIFA Ballon d'Or holder sat out last Saturday's 4-0 rout of Real Sociedad with a knee complaint, an injury that meant he could only be named among the substitutes in Tuesday's UEFA Champions League clash with Borussia Dortmund.
Real progressed to the semi-finals 3-2 on aggregate despite a 2-0 defeat in Germany in the absence of top scorer Ronaldo.
And the Portugal captain will now definitely miss Wednesday's Copa del Rey final with rivals Barcelona after Real confirmed on Thursday that he has sustained a hamstring problem.
A statement on the club's official website read: "After the tests conducted on player Cristiano Ronaldo, a muscular injury to his left hamstring (has been detected). Pending developments."
Although Real have not put a timescale on Ronaldo's return, reports suggest he will be out for around 15 days, which means he is also likely to be sidelined for the first leg of Real's Champions League last-four tie, which will take place on April 22 or 23.
However, the 29-year-old should recover for the second leg and Real's La Liga title run-in.
Ronaldo has been instrumental to Real's success this season, topping the scoring charts in La Liga with 28 goals, while also equalling the record for the number of goals in a single Champions League campaign with 14.
Real currently trail Liga leaders and city rivals Atletico by three points with six games remaining, and will face Bayern Munich, Chelsea or Atletico in the Champions League semi-finals, with the draw for the last four set to take place on Friday.
