Pineda began the regular season on the disabled list with tendinitis after throwing with diminished velocity throughout spring training. He had not thrown in a game until Saturday.
"Not good," manager Joe Girardi said of the news.
Pineda -- who joined the Yankees in a blockbuster offseason trade that sent top prospect Jesus Montero to Seattle -- felt weakness in the back of his shoulder again after throwing just 15 pitches in Tampa on Saturday.
Yankees general manager Brian Cashman said in an email that Pineda will undergo an MRI Monday in New York.
The Yankees were proceeding slowly with Pineda's rehab, limiting the 6-foot-7, 260-pound righty to bullpen sessions. On Saturday, they let him loose, hoping to see glimmers of the 94-plus mile per hour fastball that was crucial to his success as a rookie in 2011.
Pineda, 23, was 8-6 with a 3.03 ERA in the first half of last season and earned a spot in the All-Star Game as a rookie.
However, Pineda's performance dipped in the second half, going 1-4 with a 5.12 ERA.
Saturday's setback raises the question of how much Pineda will contribute in 2012.
"I think it is somewhat significant just because you were expecting him to build up," Girardi said. "Now, you are going to have to give him a little bit more time than we did before. I can't tell you when we will get him back now."
If the MRI doesn't show serious damage in the shoulder, Pineda still likely will need a full spring training-like routine to be ready to join the major league club. That would eliminate Pineda from contributing until at least the All-Star break.
While Pineda suffered a setback, Andy Pettitte was progressing so well that he may only need two more starts before he is ready for the majors. Pettitte's next start will be Wednesday for Double-A Trenton. If all goes well, he could appear in a Yankees game by about May 10.
Pineda and prospect Jose Campos were acquired for Montero and starter Hector Noesi in one of the biggest trades of the offseason.
Entering Saturday, the 22-year-old Montero was hitting .261 with two homers and seven RBIs for Seattle. Noesi, 25, was 1-2 with a 9.49 ERA. Campos had impressed in Class A, going 3-0 with an 0.58 ERA in 16 innings.