The U.S play in Antigua and Barbuda on Friday and then host Guatemala in Kansas City on Tuesday as they seek a place in the final qualifying round from the CONCACAF region for the 2014 finals.
Donovan, the national team's all-time leading goal-scorer, picked up the knee injury playing for his Major League Soccer team L.A. Galaxy against Real Salt Lake on Saturday.
The forward skipped practice on Monday while his knee was evaluated by medical staff from the U.S. Soccer Federation and on Tuesday was ruled out.
FC Dallas winger Brek Shea, who was suffering from an abdominal strain and was unlikely to feature, has also been sent back to his club for further treatment.
"It's unfortunate for Landon and Brek that they won't be able to play," said U.S. national team coach Jurgen Klinsmann.
"Landon was very optimistic over the weekend when his knee was feeling much better, but now he needs time to recover. We knew Brek was a question mark coming in, and now we have a clear picture of where he stands."
No replacements were immediately named.
Just 14 players took the field on Tuesday for practice with other players doing gym work after travelling to Florida from their club action at the weekend.
Klinsmann's team are currently level on seven points in Group A with Jamaica and Guatemala - just two teams will go through to the round of six next year which will produce three automatic qualifiers for the finals in Brazil.
