Perisic could miss up to nine months depending on how his rehabilitation goes.
The 34-year-old could also have played his last game for Spurs, given his contract expires at the end this season.
Perisic joined Tottenham as a free agent after leaving Inter Milan last summer and has made 50 appearances for the club, scoring one goal and providing 14 assists.
A club statement read: “We can confirm that Ivan Perisic has suffered a complex Anterior Cruciate Ligament (ACL) injury in his right knee.
“The experienced Croatia international sustained the injury in non-contact training and will undergo surgery.
“Wishing you well in your recovery, Ivan.”
With the European championships coming up next summer, Perisic faces a race against time to be fit for Croatia.
He has won 129 caps for his country and has silver and bronze medals from the last two FIFA World Cups.
