Trainer Todd Pletcher said he's pulling the 3-year-old colt out of next weekend's Derby due to an "issue" in his left front leg.
Eskendereya stamped himself as the horse to beat after romping to wins in the Fountain of Youth and the Wood Memorial by a combined 18@1/4 lengths.
He was scheduled to work at Churchill Downs on Sunday morning, but Pletcher opted to keep the horse in the barn while sending his other Derby entries out on the track.
Pletcher said he detected something was "off" with the horse when he galloped on Saturday morning and noticed a large amount of swelling in the left front leg between the ankle and the knee when he arrived at the barn on Sunday.
"We were still kind of hoping for a miracle overnight and we didn't get it," Pletcher said. "It's the worst of timing."
Pletcher stressed that "the horse is not lame" and he's hopeful the injury, which he likened to a sprained ankle in a human, isn't career-threatening. The horse will undergo a thorough examination soon, but Pletcher said he'll speak to owner Ahmed Zayat before making any decision.
"It's a tough one," said Pletcher.
Pletcher said he spoke to Zayat early Sunday morning and said Zayat "took it better than I would have."
"He says the horse comes first," Pletcher said. "He loves Eskendereya. He's not going to do anything to take any chances. ... It's not a phone call I wanted to make."
Eskendereya was considered Pletcher's best shot at ending his 0-for-24 mark in the Derby. The Eclipse Award winning trainer still has a handful of Derby horses, but admits he believed Eskendereya had a shot at winning the Triple Crown.
"I don't think we've ever had one at this stage of his development that was that good," he said. "It's the first time I felt like we've gotten here with a horse that could withstand the three-race series."
With Eskendereya out, Lookin At Lucky is likely to become the morning line favorite. Last year's 2-year-old champion has won six times in eight career starts, but is coming off a third-place finish in the Santa Anita Derby.
Trainer Nick Zito likened Eskendereya to Big Brown, who won the Derby and the Preakness two years ago before faltering in the Belmont.
"You've got to have luck in this game though," said Zito, who will saddle Ice Box and possibly Jackson Bend in the Derby.
Eskendereya is the second straight Wood Memorial winner to be pulled from the Derby. I Want Revenge was the morning line favorite in last year's Derby, but was scratched the day of the race due to a soft tissue injury.
Pletcher shot down questions that the horse has been dealing with a leg issue for a while. Questions arose that the horse was hiding something when he wore wraps on his front legs in the Wood.
"I think his Wood performance speaks for itself," he said.
The news probably opens the door for filly Devil May Care - also trained by Pletcher - to enter the Derby. Pletcher said jockey John Velazquez, who was scheduled to ride Eskendereya, could get the mount if Devil May Care enters the race.