"It's my understanding from Secretary (Mark) Esper that you requested my resignation ... as you have requested, I am providing resignation effective February 28, 2020," Rood wrote in the letter to Trump.
"I would like to thank John Rood for his service to our Country, and wish him well in his future endeavors," Trump tweeted on Wednesday.
Rood warned against blocking U.S. military aid to Ukraine last year, and CNN reported that Rood broke with the administration on several issues in addition to the handling of aid to Ukraine, leading to a loss of support from the top.
One official told CNN that Rood was skeptical about peace talks with the Taliban as well as the administration's decision to scale down military exercises with South Korea during talks with Pyongyang.
Foreign Policy quoted sources as saying that President Donald Trump lost confidence in Rood after a steady wave of departures of senior policymakers from the Pentagon over the last year.
The decision to oust Pentagon's top policy official was based on a recommendation by National Security Adviser Robert O'Brien, according to Foreign Policy.
Rood, a former senior vice president of Lockheed Martin, has been serving as undersecretary of defense for policy since January 2018.