Zimmerman will undergo an MRI on Wednesday morning, Nationals manager Matt Williams said after the team's 7-4 win over the Rockies.
"It's concerning," Williams said. "Any time you have to leave the game with a hamstring injury, its concerning."
Zimmerman appeared to grab at the hamstring just before he reached first base, awkwardly tumbling over the bag just ahead of the throw.
Williams and Nationals head athletic trainer Lee Kuntz hurried out to the fallen third baseman and walked with him as Zimmerman slowly limped off the field, careful not to apply too much weight to the injured leg.
Zimmerman's hustle to the bag resulted in a run, with Anthony Rendon crossing the plate to cut the Colorado lead to 4-3.
The Nationals tied the score later in the inning on a single by Wilson Ramos, but the immediate concern was with Zimmerman, who entered Tuesday's game hitting .286 with five home runs and 35 RBIs in 52 games.
Zimmerman, through a team official, politely declined to discuss the injury until Wednesday, when he figured to have more information on his prognosis. Williams said the third baseman was "pretty sore" after the game.
Zimmerman missed 44 games earlier this season with a fractured right thumb, and he has occasionally dealt with a troublesome right shoulder, which was operated on in 2012.
His teammates were left hoping for the best after Zimmerman's most recent injury.
"He's a guy just coming back from the [disabled list] and was getting his swing back, so you hate to see that," said Adam LaRoche, who hit a three-run home run in the seventh. "We'll cross our fingers here and hope it's nothing major."