The Valencia ace bruised his right thigh during training and the medical staff suggested he and forward Sergio Garcia be rested for Spain's final EURO 2008- warm up game.
"Villa is the more worrying injury of the two," team doctor Jorge Candel told reporters. "He's got an injury in a muscle that shouldn't cause too much of a problem but we will have to see how it evolves over the next few days."
Midfielder Andres Iniesta will also miss the game against USA after recovering from a bout of gastroenteritis.
"It must have been something I ate when we were given a day off," said Iniesta.
On the other hand, coach Luis Aragones will be glad to welcome back star striker Fernando Torres who shook off a minor ankle strain.
Spain play in Group D of the EURO 2008 alongside champions Greece, Sweden and Russia.