European Commission President Jose Manuel Barroso said states breaking away from existing EU countries would struggle to gain EU membership, further complicating Scottish nationalists' already uncertain plans for independence.
Barroso said in an interview on Sunday it would be nearly impossible for the European Union to grant membership to such states - days after the British government said an independent Scotland would not be able to keep sterling as its currency.
Scotland is due to hold a referendum on independence in September. Polls show around 29 percent of voters in favour and 42 percent against, with 29 percent undecided.
Barroso, interviewed on BBC television, declined to comment directly on whether an independent Scotland would be welcome to join the EU.
But he said all EU states would need to back the membership of any new country that emerged from a current member state.
"It would be extremely difficult to get approval of all the other member states ... I believe it's going to be extremely difficult, if not impossible," he said.
The Scottish National Party (SNP), which is fronting the independence campaign, is banking on retaining both EU membership and the pound.