The 49-year-old actress is said to have told friends she has "had enough" following a string of allegations against the 'Pulp Fiction' star of sexual misconduct from a number of male masseurs, and the publication of pictures the star dressed as a woman at a private event in 1997.
A source told the National Enquirer magazine: "Kelly told me their marriage is over and made it clear that she's no longer living in the family home.
"She's put up with John's double life for decades while his behavior remained in the shadows.
"But all this coming out in public has humiliated her. Kelly is absolutely destroyed."
The photographs, which were obtained by National Enquirer, show the 58-year-old actor at a party in Los Angeles wearing a white dress with a bra visible underneath, a brown belt, fur stole, earrings and a necklace, and wears eyeliner, pink lipstick and a blonde wig.
John's lawyer Marty Singer has strongly denied the claims from four masseurs that the actor behaved inappropriately when they visited him at separate times.
John and Kelly - who married in 1992 - have children Ella, 12, and 18-month-old Benjamin together.
They also had a 16-year-old son, Jett, who tragically died in January 2009.