The Spanish wing-back is set for surgery on his right knee after limping off early on in the Europa League last-16 win against Olympiacos on Thursday.
It means he will play no part in Germany as Wolves look to go all the way in the competition, and he is set to miss a large chunk of next season as well. ACL injuries tend to take six to nine months to recover from following an operation.
Otto left Molineux on crutches on Thursday night, and their worst fears were confirmed when he was assessed on Friday.
Wolves doctor Matt Perry said: “In the first-half of the match with Olympiacos, Jonny suffered an injury to his right knee.
“A scan confirmed the first clinical impression of an injury to the anterior cruciate ligament, which will require surgical repair.
“This has been arranged and the whole team wish Jonny well on his path to recovery.”
With Jonny unable to play any further part in the Europa, young centre-half Lewis Richards will travel with the squad to Germany tomorrow, ahead of Tuesday's semi-final against Sevilla in Duisburg (8pm UK time).
It is a big blow as Otto has been one of Nuno Espirito Santo's most-trusted troops ever since arriving in the summer of 2018, initially on loan before he was signed permanently from Atletico Madrid for £15million.
In total, the 26-year-old has made 87 appearances in gold and black, scoring three goals along the way.
Richards, meanwhile, has not yet played for the first team, but the 18-year-old has impressed for the under-23s over the past year. He won the academy player of the year award last season.