The president of the European Commission has said it would be "very difficult, if not impossible" for an independent Scotland to join the EU.
Talking to Andrew Marr, Jose Manuel Barroso said the country would have to reapply for membership and secure agreement from all member countries.
Barroso highlighted the likelihood of Spain scuppering such a vote in order to not give legitimacy to its own separatist movement in the Catalan region.
Despite saying he was trying to avoid stepping into the conversation around independence, his comments were described as a "hand grenade" being thrown into the debate.
He said: "In case there is a new country, a new state, coming out of a current member state, it will have to apply and... the application and the accession to the European Union will have to be approved by all the other member states of the European Union.
"I don't want to interfere on your referendum here, your democratic discussion here, but of course it will be extremely difficult to get the approval of all the other member states to have a new member coming from one member state.
"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."
He called on the prime minister to distance himself from "astonishing" claims by a party source that a 'Yes' vote would not automatically mean Scotland becomes independent.
It has been a tough week for Salmond after all three of the major parties united in claims that an independent Scotland would not be able to keep the pound.
George Osborne said: "If Scotland walks away from the UK, it walks away from the UK pound.
"I could not as chancellor recommend that we share the pound with Scotland.
"The evidence shows that it would not work and it would cost money and it would not provide economic security for the rest of the United Kingdom."
The Scottish Government has previously said it will negotiate Scotland's EU membership in the 18 months after the referendum.
Scottish ministers want the negotiations to be hammered out from within the Union while Scotland remains part of the UK, according to the Government's White Paper on independence.
The independence referendum will take place on September 18, with voters being asked: "Should Scotland be an independent country?"
Scotland's Deputy First Minister Nicola Sturgeon branded Mr Barroso's stance "preposterous".
Ms Sturgeon said: "This is a preposterous assertion - as the ridiculous comparison with Kosovo illustrates. 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.
"The fact of the matter is that no member state has said that it would veto Scotland's continuing membership.
"We have put forward a suggested mechanism by which Scotland can make the transition from being in the European Union as part of the UK to being part of the European Union as an independent country.
"This can be done with continuity of effect and no detriment to other European states.
"And as a number of experts, including former European Court judge Sir David Edwards and honorary vice president of the European Commission Graham Avery, have said this is a process that will take place in the period between a vote for independence and Scotland becoming independent.
"The question Mr Barroso was asked was would Scotland be welcome. Every indication we have is that the answer to that question is yes.
"Indeed, the only threat to Scotland's membership of the EU is if we don't become independent and face the prospect of David Cameron's in/out referendum in 2017."