Reps for Chalke, 46, confirm to PEOPLE that she and fiancé Jamie Afifi "separated some time ago."
"They remain committed to being devoted co-parents and good friends," the reps add of the Scrubs actress and the entertainment lawyer.
Chalke and Afifi, 49, got engaged in 2006, though the pair never tied the knot in the years that followed. They have two children together — Charlie, 12, and Frances, 6.
In 2017, Chalke told PEOPLE she didn't ever expect to be a boy mom.
"When I initially found out I was pregnant the first time, I just assumed it was going to be a girl because I come from all girls," she said. "I was thinking, like, 'A boy? What do you do with a boy? I don't know how to do that.'"
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Now, she "can't imagine not having gotten to parent a boy." She added, "It's Lego structures and lightsaber fights, water balloon fights after school and ice hockey."
The Scrubs alum added that her two kids are "very different human beings."
On the horizon for Chalke is season 2 of Netflix's Firefly Lane, set to premiere later this year.
Alongside Katherine Heigl, Chalke plays one half of a best friend duo as they navigate changes and traumas throughout their lifespan. The stakes will be high in season 2 as the season 1 finale ended with the lifelong friends at odds over a mysterious feud.
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During a Zoom call with fans in May 2021, Chalke teased the reason for the fight.
"So, this issue between Tully and Kate, we can't say much, but we can say that it's family related," she said. "And it has nothing to do with Johnny [Ben Lawson]."
In addition to Firefly Lane, Chalke will once again voice the character of Beth Smith on Adult Swim's series Rick and Morty. The fifth season premiered on June 20.