Rodriguez will undergo an MRI on Wednesday.
"I'm pretty sore," said Rodriguez, who declined to speculate as to whether he would go on the disabled list.
On a fifth-inning double-play groundout, Rodriguez pulled up prior to first base just before the throw beat him. Immediately afterward, he looked as if he grabbed his hamstring. Rodriguez walked off gingerly and mixed a little slow jogging into his trip off the field.
"It is definitely worrisome," Yankees manager Joe Girardi said.
In the eighth inning, when the DH's next turn came up, Dustin Ackley pinch hit for Rodriguez.
Rodriguez missed two games last week because of an oblique injury. For the struggling Yankees, he has been a recent bright spot in the lineup. He entered Tuesday having gone 6-for-14 in the Yankees' previous six games. Even so, he began the game batting just .203 with five homers and 12 RBIs.
"It is extremely frustrating," Rodriguez said.
Rodriguez, 40, is being paid $21 million this season and is owed $21 million more for 2017, which will be the final year of his 10-year, $275 million contract.
If Rodriguez were to go on the disabled list, the Yankees could possibly call up Nick Swisher to replace him.