Jeter came out of New York's 5-3 victory over Washington on Tuesday night after seven innings. His MRI on Wednesday was fine, but Girardi opted to keep him in the dugout during New York's 3-2 loss to the Nationals.
"It worries me a little bit, but he told me he was playing tonight," Girardi said before the game, "which makes me believe that he feels better. My thought process, though, is if he goes out there and he just irritates it again then we're back at square one."
Jeter said his ankle was sore before the series opener against Washington and got worse during the game, but downplayed the ailment in his typical fashion.
"It wasn't a particular play where I felt something," he said. "I think it just progressed but it's not a big deal."
Pena replaced Jeter when he left Tuesday's game and hit an RBI single in the eighth inning.
Jeter said he might have hurt himself Sunday when Gary Sheffield of the New York Mets slid into second base during a double play, but he wasn't certain. The nine-time All-Star is batting .306 with nine homers and 30 RBIs in 61 games this season.
"Just banged up a little bit," Jeter said.
Also on Wednesday, injured outfielder Xavier Nady (right elbow ligament) threw in front of Girardi and said he's hoping to play four innings in the field in a rehab game in Florida on Friday.
"Hitting's fine. I haven't had any troubles hitting," Nady said. "It's just obviously throwing. But the last few days have gone good, so that's encouraging and I feel like each day it's kind of progressing."