The Scottish FA confirmed the news in a statement.
"We regret to announce that the knee injury sustained by Billy Gilmour in today's win over Curaçao will rule him out of participation in FIFA World Cup 2026. Everyone involved with the Scotland men's national team wishes Billy a speedy recovery. He will now return to his club, SSC Napoli, to commence rehabilitation."
Scotland head coach Steve Clarke was unambiguous about the scale of the loss.
"I am devastated for Billy because he has been an integral part of our World Cup qualifying campaign. The timing of this injury is so, so cruel and we all feel for him. He knows what we all think of him as a footballer and as a person, and while no words will give him any comfort tonight, I am certain Billy will have many major tournaments ahead of him in the future."
Napoli midfielder Gilmour, 24, had made 46 international appearances for Scotland and was one of the most important members of Clarke's squad. He limped off during the first half of the Curaçao match and was taken directly from Hampden for a scan.
His replacement may already have presented himself. Manchester United midfielder Fletcher, 19 — son of former Scotland captain Darren Fletcher — was not in Clarke's 26-man squad but joined the camp this week to make up training numbers ahead of the Curaçao game. He was introduced at half-time and produced an accomplished 45-minute display in his senior international debut.
Clarke, who flies out to the United States with his squad on Sunday, acknowledged the Fletcher option directly but was careful not to commit.
"Obviously I've got Connor Barron, Andy Irving, Lennon Miller, and you can see for yourself how good young Tyler is. The three I mentioned are on standby. Tyler joined us this week and trained well, so he is a little bit closer than the other three. But I would need to have a big discussion with my staff before deciding that's the way to go."
He added: "Tyler — okay, against ten men — showed that he has the capabilities of being a big player for us in the future. The players are impressed, the coaching staff are impressed."
Ross Stewart is also carrying a knee problem and was absent for the Curaçao match. Clarke confirmed he will train in the United States next week with the Bolivia friendly in New Jersey on Saturday his target to return.
Scotland open their World Cup campaign against Portugal in Group K.