The 29-year-old overcame a hamstring complaint to play in Real Madrid's Champions League final victory over Atletico Madrid, but linked up with his international team-mates on 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.
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 prepare for their latest pre-World Cup friendly against Greece on Saturday.
None of these players are believed to be serious doubts 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 play-off with Sweden to almost single-handedly fire Paulo Bento's men to Brazil.
Portugal will need him at his brilliant best if they are to advance from a tough group containing Germany, USA and Ghana.
Their World Cup campaign kicks off with a clash against Germany on June 16.