Crosby was skating during the pregame practice when he said he felt discomfort in the ankle. He recently missed 21 games with a sprain in that ankle.
Crosby had two goals Sunday against Washington in a 4-2 victory, his first goals in three games since returning from the injury, which he suffered Jan. 18.
Crosby resumed playing March 11 in Tampa, setting up Maxime Talbot's winning goal with about 2½ minutes remaining in a 2-0 victory.
Crosby has had discomfort in the ankle since the game against the Capitals.
"That game itself was pretty physical," Crosby said. "It's probably a combination of that and it's been sore the past couple of days."
The Penguins have three days off following Wednesday's game, and coach Michel Therrien said it made sense for Crosby to sit out and rest during the break. The Penguins next play Sunday at home against Philadelphia.
"Obviously, it's a little sore now," Crosby said. "We have a few more days until our next game. If there is a time to give it a rest, it might be now ... It's one of those things where you might not get three or four days [off] again in the schedule."
Asked if he hoped to play Sunday, Crosby said, "That's what I'm planning on."
Crosby, last season's NHL MVP, has 22 goals and 45 assists for 67 points in 49 games.
Talbot also will sit out Wednesday after aggravating the high ankle sprain he suffered earlier this season.
Penguins goalie Marc-Andre Fleury also was out nearly three months with the same injury before returning Feb. 28.
A high ankle sprain can flare up at any time -- Talbot has had several setbacks since getting hurt Nov. 3 -- and often lingers for months.
"It's just the nature of it and you have to live with it," Crosby said. "It's not fun, for sure."