Pato came on as a substitute in the Champions League match against Barcelona on Tuesday but lasted only 13 minutes before limping with another injury.
Milan, who last month sent the player to the United States to try and clear up his constant injury problems, confirmed that Pato had suffered a problem in his left thigh and would undergo further tests in 10 to 15 days.
"AC Milan wishes to communicate that Alexandre Pato underwent medical scans today at the Galeazzi Orthopedic Institute in Milan," said the club in a statement.
"The scans revealed a muscular lesion in his left thigh. Between 10 and 15 days from today, the player will undergo more medical tests."
With the season due to finish in mid-May, Italian media concluded that he would take no further part in Serie A where Milan have a two-point lead over Juventus.
Pato, full name Alexandre Rodrigues da Silva, was seen as one of the Brazilian hopes for the future when he burst onto the scene with Porto Alegre club Internacional in 2006.
Pato means duck, although he takes the name as he was born in the town of Pato Branco in southern Brazil.
He was included as a 17-year-old in their squad for that year's Club World Cup in Japan and scored in the 2-1 win over Egyptian side Al Ahli, making him the youngest player to score in a FIFA competition.
He joined AC Milan in August 2007 but was only able to play competitive games for them the following January, the first transfer window after his 18th birthday.
Hopes were raised further when he marked his Brazil debut with a stunning goal in a friendly against Sweden the following March.
But his injury troubles began in the 2009/10 season, costing him a place in Brazil's World Cup squad.
Since then, he has played well in fits and starts. He scored two goals for Brazil at the Copa America last year but failed to inspire them beyond the quarter-finals where they lost on penalties to Paraguay.