Chasing Dreams, ante-post favourite for the Queen Mary Stakes at Royal Ascot next week, will miss the race.
Charlie Appleby's filly made a blistering start to her career at Newmarket in April when winning by five lengths.
The runner-up that day, David Evans' Good Vibes, has subsequently won both her races - including the Listed Marygate Stakes at York.
As low as 3-1 with some firms for the Group Two race, Chasing Dreams was found to be lame on Thursday.
In a tweet, her owners Godolphin posted: "Chasing Dreams, an intended runner in the Queen Mary Stakes at #RoyalAscot next week, was found to be lame this morning and unfortunately will no longer be taking up that engagement."