Nancy Pelosi has said she was ordered to leave her office in the US Capitol by the new interim House Speaker Patrick McHenry.
The request came just hours after Mr McHenry was appointed acting Speaker. His Republican ally Kevin McCarthy was ousted from the role on Tuesday.
Mrs Pelosi criticised the move, saying in a statement that she had been asked to leave immediately and "this eviction is a sharp departure from tradition".
Mr McHenry has not yet commented.
While it is rare for members of Congress who are not in leadership roles to have offices in the Capitol building, Mrs Pelosi, a Democrat, was able to keep one as she is a former House Speaker herself.
"Sadly, because I am in California to mourn the loss of and pay tribute to my dear friend Dianne Feinstein, I am unable to retrieve my belongings at this time," she said in the statement.
Ms Feinstein, a California senator who served for more than three decades, died aged 90 last week.
"Office space doesn't matter to me, but it seems to be important to them," Mrs Pelosi added.
The request for Mrs Pelosi to leave her office was first reported by Politico, which is among several US media outlets to have viewed the email apparently sent to her staff from the House Administration Committee.
"The Speaker pro tempore is going to re-assign H-132 for speaker office use. Please vacate the space tomorrow, the room will be re-keyed," the email reportedly reads.
A source familiar with the matter told NBC News that the decision was made by the previous Speaker's office. Mr McCarthy has not commented.
Mr McHenry assumed the role after Mr McCarthy became the first ever House Speaker to lose a no-confidence vote on Tuesday. He was toppled after a a right-wing revolt from within his own party.
While the 47-year-old has taken on the role on an interim basis, there is no clear successor to oversee the slim House Republican majority.
The House is now in recess for a week with a potential vote on electing a new Speaker likely next week.