The Prime Minister seized on comments from the European Commission on Monday which indicated that an independent Scotland would have to apply to join the EU, rather than automatically being a member.
Commission spokesman Margaritis Schinas said the “Barroso doctrine” continued to apply.
Former commission president Jose Manuel Barroso set out the legal view that if one part of an EU country became an independent state it would have to apply for EU membership.
At Prime Minister’s Questions, Mrs May said: “Scotland will be leaving the European Union, it will leave the European Union either as a member of the United Kingdom or were it independent. It’s very clear with the Barroso (doctrine), it would not be a member of the European Union.
“What we need now is to unite, to come together as a country and to ensure that we can get the best deal for the whole of the United Kingdom.”