Barroso's blunt statement ramps up the pressure ahead of Scotland's key referendum on independence from the United Kingdom, following Britain's warning that it will not be allowed to keep the pound if it votes to break away.
"I believe it's going to be extremely difficult, if not impossible" because the formation of a new member from a member state needs approval from all other EU nations, Barroso told BBC television.
Scots are due to vote on independence on September 18 in a historic referendum that could result in the breaking up of its 300-year union.
"We have seen Spain has been opposing even the recognition of Kosovo, for instance. So it is to some extent a similar case because it's a new country and so I believe it's going to be extremely difficult, if not impossible, a new member state coming out of one of our countries getting the agreement of the others."
The comments drew a sharp rebuke from Scotland's Deputy First Minister Nicola Sturgeon, whose Scottish National Party (SNP) is spearheading the pro-independence campaign.
"This is a preposterous assertion -- as the ridiculous comparison with Kosovo illustrates," Sturgeon said in a statement. "Scotland is already in the EU and has been for 40 years.
"As Mr Barroso rightly says, the question of Scotland's independent membership of the EU is a matter for the democratic wishes of the people of Scotland and the views of other member states -- not the European Commission," she said.
Britain, which is campaigning for a "no" vote in the referendum, has already warned that an independent Scotland would have to leave the pound.
Finance minister George Osborne said on Thursday that there was "no legal reason" why the rest of the United Kingdom would have to share the pound.
All three of Britain's three main political parties argue that they will not let an independent Scotland retain the pound sterling currency.
The Scottish government has previously said it will seek to negotiate Scotland's EU membership in the 18 months after the referendum, should Scots vote in favour of leaving the UK.
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