The actor, who played Chandler Bing in the hit sitcom, had been dating the literary manager since 2018, with the couple becoming engaged last November.
Matthew revealed he and Molly have now parted ways in a statement.
He told People: “Sometimes things just don’t work out, and this is one of them. I wish Molly the best.”
The couple previously split briefly last year before reuniting, with Matthew later proposing.
Announcing his engagement at the time, he told People: “I decided to get engaged.
“Luckily, I happened to be dating the greatest woman on the face of the planet at this time.”
Matthew was previously in a relationship with Masters of Sex actor Lizzy Caplan, who he dated for six years before their split in 2012.
News of Matthew and Molly’s split comes less than a week after he was seen getting back together with his former Friends castmates on the highly-anticipated reunion.
Following its debut on both sides of the Atlantic last Thursday, director Ben Winston spoke out against “unkind” comments made about Matthew online after some people questioned whether the actor was okay, noting how rarely he spoke during the show.
“People can sometimes just be unkind. I wish they weren’t,” Ben told The Hollywood Reporter. “I loved working with him. He’s a brilliantly funny man and I thought he had some great one-liners in the show.
“I felt just happy and lucky to be in his presence and directing him on something like this.”
Echoing those sentiments, Friends producer Kevin Bright told The Hollywood Reporter in a separate interview that it “was great seeing” Matthew and “what people say is what people say.”
“I think he’s OK. He seems stronger and better since the last time I saw him, and excited about going forward,” he shared.