The picture shows Paul McCartney, John Lennon, George Harrison and Ringo Starr walking across the iconic zebra in the opposite direction to that on the classic album cover for Abbey Road.
The 'backwards' photograph is expected to fetch up to £9,000.
The photo sees the legendary band walking right to left, while McCartney is wearing sandals instead of the bare feet that he had in the original 1969 image.
It is one of 25 prints being sold by a private collector at Bloomsbury Auctions next week (May 22), with viewings beginning on Friday (May 18).
Only six photographs were taken on the day by Iain Macmillan, taking just 10 minutes with a police officer holding up traffic.
Macmillan, who died in 2006, once said of the photos: "The one eventually chosen for the cover was number five of six. It was the only one that had their legs in a perfect 'V' formation.
"That photo's been called an icon of the 1960s. I suppose it is. I think the reason it became so popular is its simplicity. It's a very simple, stylised shot. Also it's a shot people can relate to. It's a place where people can still walk."
The zebra crossing on Abbey Road, St John's Wood has been given Grade II listed status.
Many Beatles fans visit the iconic site, with a permanent webcam of the zebra crossing viewable online.