Group One winner Commanding Jewel will undergo scans on Tuesday to determine her spring fate.
Trainer Leon Corstens ruled Commanding Jewel out of Sunday's Group One Sir Rupert Clarke Stakes on Monday with the three-quarter sister to Atlantic Jewel appearing to have a hamstring issue.
"As she trotted off this morning she didn't seem quite right in the near hind and on pulling up it was the same," Corstens said.
"I got the vet to have a look at her and the vet said it looks like she's pulled a muscle behind.
"We're going to scan it tomorrow, just evaluate it and work it out from there."
Commanding Jewel was to have her next start in the Rupert Clarke Stakes at Caulfield after winning the Group Two Let's Elope Stakes second-up after almost a year off due to a joint injury.
"I treated her so she's definitely not running on Sunday," Corstens said.
He said he was hoping it wasn't the end of the mare's spring carnival campaign.
"I don't know. I would like to say definitely no, but you never know with these sorts of things," the trainer said.
"I couldn't work her fast tomorrow so there was no hope of her running on Sunday.
"If they are not fit, you can't win races."
Commanding Jewel won last year's Group One Thousand Guineas (1600m) and Damien Oliver was due to reunite with the mare in the Rupert Clarke Stakes having not ridden her in a race since.
The four-year-old daughter of Commands missed the autumn and resumed with a sixth in the Listed Carlyon Stakes (1000m) before returning to the winner's circle in the Let's Elope.