The 24-year-old came on for the final 33 minutes of Atletico Madrid’s 1-0 Champions League semi-final second leg defeat to Arsenal on Tuesday (May 5).
Cardoso suffered a "high-grade" right ankle sprain during training two days later, putting his chances of playing at the World Cup in major doubt.
"Johnny Cardoso was unable to complete Thursday's training session on May 7 after suffering a right ankle injury," a statement from Atletico Madrid read.
"Medical staff conducted tests, and the medical report indicates that the midfielder has a high-grade sprain of his right ankle.
"The Rojiblanco number '5' will undergo physiotherapy sessions and rehabilitation work in the gym, and the evolution of his injury will determine his return to competition."
Though the severity of the injury isn’t yet known, it’s expected Cardoso will be out for around five weeks, with the USMNT set to release its World Cup squad on May 26.