Mr Mahathir's shock resignation comes amid rumours that he may form a new coalition without his designated successor, Anwar Ibrahim.
The world's oldest prime minister came into power in 2018.
In a shock victory, he ousted then-prime minister Najib Razak, who has been linked to a multibillion-dollar scandal involving a government fund.
The Prime Minister's Office said the letter of resignation was submitted at 13:00 local time (05:00 GMT). No other details were included in the statement.
It is not clear who will be the next prime minister or if new elections will be called.