The 29-year-old overcame a hamstring complaint to play in Real Madrid's Champions League final victory over Atletico Madrid, but joined up with his international teammates Thursday carrying a fresh injury.
The forward was one of five players named on Portugal's official website that were forced to remain on the sidelines — four of whom were nursing thigh injuries.
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A brief statement said Ronaldo was suffering from "myalgia (pain) in the posterior region of his left thigh" and underwent specific rehabilitation work at the team hotel.
He was joined there by Raul Meireles, Hugo Almeida, Rubon Amorim and Joao Moutinho, as Portugal prepares for a pre-World Cup friendly against Greece on Saturday.
None of these players is believed to be a serious doubt for the tournament, though there will be concerns over Ronaldo given the Seleccao's reliance on the forward during qualification.
The reigning Ballon d'Or winner scored a hat trick in the second leg of their World Cup playoff with Sweden to almost single-handedly fire Paulo Bento's men to Brazil.
Portugal will need him at his best if it is to advance from a tough group containing Germany, the USA and Ghana.