Neither the Pentagon nor the White House offered a public explanation.
In his resignation letter addressed to the president and dated Feb. 19, Rood wrote, "It’s my understanding from Secretary Esper that you requested my resignation from serving as Under Secretary of Defense for Policy. Senior administration officials appointed by the President serve at the pleasure of the President, and therefore, as you have requested, I am providing my resignation effective February 28, 2020,” his letter said.
In a tweet on Wednesday, Trump thanked Rood for service to the country and wished him well in future endeavors.
Rood became linked to the impeachment inquiry into the president in part because he was the senior defense official who certified in a letter to Congress in May that Ukraine had made "sufficient progress" towards defense and corruption reform that allowed the $250 million in security assistance funding to flow.
The next month, that aid was withheld by the White House in violation of the law, according to the Government Accountability Office, with Trump administration officials testifying during the impeachment hearings that it was part of a quid pro quo at Trump's request where aid would only be released if the Ukrainian government agreed to investigate former Vice President Joe Biden and his son Hunter.
Rood told reporters in December that he became aware that military aid to Ukraine had been held up "significantly after May" but "never received a very clear explanation" as to why.
Asked by ABC News if there was a link between Rood's resignation and his role in certifying the aid to Ukraine, Chief Pentagon spokesperson Jonathan Hoffman said during a news conference on Wednesday that sounded "speculative."
"I have no information that would lead me to that conclusion," Hoffman said.
He could not say whether Esper recommended to the president that Rood be removed and referred back to the resignation letter.
A defense official told ABC News that Rood's resignation should not be linked to the impeachment inquiry. A former official said the departure is more likely because of Rood's policy disagreements over issues like Syria, North Korea, Iran, and Ukraine.
However, Rood's divisive leadership style has been reported as a source of frustration within the department. In December, Foreign Policy reported that many current and former defense officials blamed Rood for creating a toxic work environment and identified him as a reason the Pentagon saw an exodus of top officials and was struggling to fill posts.
"I would like to thank John Rood for his service to the Department," Secretary of Defense Mark Esper said in statement released by the department. "John has played a critical role on a wide range of DoD issues including modernizing our nuclear deterrence capability, efforts to increase burden sharing by our NATO allies, our Missile Defense Review and implementing the National Defense Strategy. I wish him all the best in his future endeavors."
In that statement, Pentagon press secretary Alyssa Farah announced that James Anderson, the current senior official Performing the Duties of the Deputy Under Secretary of Defense for Policy, will take over Rood's responsibilities until a permanent replacement is appointed by the president and confirmed.
Rood wrote in his resignation letter that he was drawn to public service beginning with his time with the Central Intelligence Agency in 1988. He later served at the State Department, Pentagon, National Security Council, and as a staffer on Capitol Hill before working in the private sector at two large defense companies, Raytheon and Lockheed Martin.
"I thank you for giving me the privilege of again contributing in service to our Nation," Rood told the president.
"I leave with the utmost admiration for the outstanding team with which I worked at the Defense Department," he concluded.