Millisle came from off the pace to defeat hot favourite Raffle Prize in the Juddmonte Cheveley Park Stakes at Newmarket on Saturday.
Raffle Prize won Group Two contests at Royal Ascot and the July Festival when landing the Queen Mary and Duchess of Cambridge Stakes, while she was just touched off by Earthlight in the Group One Prix Morny.
Sent off the 10/11 favourite to win her first top-level contest under Frankie Dettori, she looked to have seen off her rivals inside the final furlong.
However, Shane Foley had held up Jessica Harrington's Millisle in the rear in the early stages and she came through with a strong run to win by a length-and-three-quarters from the favourite, with Tropbeau third.
Harrington said: "She got beat at Salisbury (Dick Poole Fillies' Stakes), she was coming back at the end but had to do all the donkey work that day. We just thought the rising ground here would suit her.
"Shane said the further she was going, the better she was going. She is bred to get a mile easily, as I actually trained a half-sister of hers that got a mile and six, so there is plenty of stamina on the dam's side.
"You would probably give her an entry (in the 1000 Guineas), but the ground would have to be good. I know she has won on soft ground, but you would have to start questioning the trip on soft ground.
"She is finished for the year now, that is her fifth run and she hasn't done badly. She broke her maiden at a very illustrious course called Bellewstown! Anyone who goes to Ireland will know where that is.
"We thought early on in the spring she would be our first two-year-old, then she started to grow and got a bit weak on me, but she showed a lot of speed early on.
"At no point did I think she was going to win until the last 100 yards."
A deflated Johnston said: "I think that is our seventh second in a Group One this year, although it could be eight. It is at least seven, so it is pretty sickening.
"Frankie said she did everything right, but in the last 50 yards that one has come from the clouds and he didn't know where she came from.
"She has not had a day off all year and there are not many horses we can say that about. She has been perfectly straightforward. That would be it for the season, as I don't think there is anything else.
"It is sickening not to have had a Group One with her. Frankie got off and said she is my Guineas filly for next year.
"That would normally be my attitude with these sort of horses, as if they don't get the trip they can come back in trip later."