European Commission President José Manuel Barroso has said it would be nearly impossible for the European Union to agree on granting membership to newly-formed states that elect to break away from an existing member country.
Mr Barroso's comments were made after he was asked if an independent Scotland would be welcome to join the EU.
However, he said he did not want to comment specifically on that scenario.
Scotland is due to hold a referendum on independence in September.
"It would be extremely difficult to get approval of all the other member states to have a new member coming from one member state," Mr Barroso said in a BBC television interview.
"I believe it's going to be extremely difficult, if not impossible."
Mr Barroso, whose mandate as head of the EU executive ends in October, has previously said that any newly independent state would have to re-apply to join the EU.
Mr Barroso's comments would appear to be another setback for Scottish First Minister Alex Salmond and his hopes for independence.
British Chancellor George Osborne declared on Thursday that he would not be prepared to enter a currency union with an independent Scotland.
Mr Salmond went on the offensive against critics of the independence campaign.
Writing in The Sunday Times, he accused the British government of bullying over the currency issue.
Mr Salmond also said he had asked British Prime Minister David Cameron to rein in his campaign to keep Scotland's union with the rest of Britain intact.