A source familiar with the situation confirmed Yovanovitch's exit on Friday.
A senior State Department official told Fox News that after filing for retirement, Yovanovitch could remain on the payroll for months, especially if she finishes her department assignment at Georgetown.
As of Friday evening, the State Department email system still directs to her Georgetown address.
Yovanovitch, who worked for the State Department for 30 years, was thrust into the public spotlight after she testified at Trump's impeachment hearings in November 2019.
Trump recalled her from her post as ambassador to Ukraine in May 2019. Despite the president's recall, she remained a State Department employee until now.
In a video released by Fox News on Saturday, Trump can be heard calling for her removal as early as April 2018, after speaking with Lev Parnas and Igor Fruman -- former Rudy Giuliani associates who have since been indicted.
Parnas and Fruman met with the commander in chief and claimed that Yovanovitch had allegedly been speaking negatively about the president.
"Get rid of her. Get her out tomorrow. I don't care. Get her out tomorrow. Take her out. OK? Do it," Trump can be heard saying on the tape.
Trump defended his decision by saying that, as president, "I have a right to hire and fire ambassadors."
When Yovanovitch testified in November 2019 about being recalled by Trump, she said she believes Giuliani played a key role in telling people she was not supportive of the president.
“I do not understand Mr. Giuliani’s motives for attacking me, nor can I offer an opinion on whether he believed the allegations he spread about me,” Yovanovitch said.
She also claimed the efforts against her by presidential allies obstructed her work.
“If our chief representative is kneecapped, it limits our effectiveness to safeguard the vital national security interests of the United States,” Yovanovitch said.
Trump fired back in a tweet that same day and said ambassadors serve the executive branch and can be removed or replaced.
"It is a U.S. President’s absolute right to appoint ambassadors," he wrote in the tweet. "They call it serving at the pleasure of the president."
Fox News' Rich Edson, Adam Shaw and John Roberts contributed to this report