Set to be seeded for the first time at his home grand slam, the world No.26 received treatment to his right calf before pulling the pin, down 6-1 2-1 citing foot pain, to defending champion Grigor Dimitrov on Friday.
Thompson enjoyed the best year of his career in 2024 and began in similar fashion this week, with fighting victories against Matteo Berrettini and Alex Michelsen to again reach the final eight in Brisbane.
But, in superb form, world No.10 Dimitrov was offering no favours as he controlled the points and left Thompson wincing as he scrambled side-to-side.
Get well soon, Thommo �— Tennis TV (@TennisTV) Defending champ @GrigorDimitrov moves through as Jordan Thompson is forced to retire, 6-2 2-1 RET #BrisbaneTennis pic.twitter.com/9TuHkGKKA9January 3, 2025
When Thompson walked to the net it marked the second straight year the rejuvenated Bulgarian had sent him packing from the tournament.
The Australian Open begins on January 12, Thompson's injury scare an unwanted headache as he readies for his first major with the protection of a seeding.
A wincing Thompson received treatment between sets and then again before what was to be the final game of the match, the trainer rubbing his lower leg but never taking off the player's shoe.
"It's very unfortunate for him, I hope he's ready for the Aussie Open," Dimitrov said.
"From the beginning I felt like I was in control ... all those things, they're giving me a lot of confidence."
Jordan Thompson reacts during a difficult outing against Bulgaria's Grigor Dimitrov. (Darren England/AAP PHOTOS)
Dimitrov will play Jiri Lehecka in one semi-final, while Novak Djokovic faces Reilly Opelka later on Friday night for a semi-final spot on the other side of the draw.
The winner of that match will face France's big-serving rising star Giovanni Mpetshi Perricard, who is yet to drop a service game on his way to the semis after beating Czech Jakub Mensik 7-5 7-6 (7-5).
Djokovic is seeking his 100th ATP title in Brisbane, the former world No.1 and winner of 24 grand slam titles boasting just a single trophy in 2024 - the Paris Olympic gold.