The Portuguese Football Federation said in a statement that the 29-year-old Real Madrid forward has been suffering from tendinitis in the patella tendon.
The FPF's medical team had spoken in recent days of the Ballon d'Or winner's thigh problem, but the knee issue will be a further worry to national coach Paulo Bento ahead of the team's opening match against Germany in Salvador on June 16.
Earlier, Real president Florentino Perez urged the national team's manager not to risk the 29-year-old if he was not fully fit.
Perez told the Spanish radio network Cadena SER: "I am concerned that any player can pick up a serious injury, as has happened [in the past] with Kaka and [Sami]Khedira.
"A player like Cristiano Ronaldo should only play if he is fit."
Ronaldo had several minor niggles during the closing weeks of the 2013-14 domestic season, but went on to play 120 minutes in the Champions League final success against Atletico Madrid.
The former Manchester United striker trained on Tuesday for the first time on grass, undergoing a specific individual programme in New York ahead of the squad's friendly against Mexico tomorrow.