Coneygree is lame and highly unlikely to take his chance in the Albert Bartlett Novices' Hurdle at Cheltenham on Friday.
The six-year-old, trained by Mark Bradstock, returned stiff after what had been a pleasing workout on Saturday.
The horse will be checked over on Monday and should the issue be found not to be serious, Coneygree will be re-routed to run at Aintree's Grand National meeting next month.
"He did the most fantastic, best-ever piece of work on Saturday. He came in a little bit stiff and last night he was lame behind," said Bradstock's wife and assistant, Sara.
"It will be investigated on Monday morning, but he's got a massive future.
"We are not going to take any risks, so it is extremely unlikely he will run (at Cheltenham).
"He is still a big weak horse and that is when they get these training injuries.
"Hopefully it's nothing serious and if it is just a muscle tweak he will probably go to that three-mile hurdle (Grade One John Smith's Sefton Novices' Hurdle) at Aintree."
Coneygree has looked a bright prospect, winning his first three races over hurdles before finishing third behind Festival hopes At Fishers Cross and The New One at Cheltenham in January.