Alex Salmond's problems worsened last night after Spain's foreign minister warned an independent Scotland would be forced to "join the queue" and win support of all 27 members before it would be allowed into the EU.
Jose Manuel Garcia-Margallo's appeared to directly contradict Mr Salmond's position following Tuesday's controversy in which the First Minister had to issue a statement denying he was a liar over the question of whether legal advice had been sought on EU membership in an independent Scotland.
Mr Garcia-Margallo told Spain's senate that after independence, Scotland would face a potentially tortuous negotiating process and would also need his country's support.
He said: "In the hypothetical case of independence, Scotland would have to join the queue and ask to be admitted, needing the unanimous approval of all member states to obtain the status of a candidate country - and to sign the final treaty [of accession]."
He suggested EU members nations would need to check carefully Scotland's legislation before approving the 35 separate chapters that have to be negotiated before admission would be granted. EC president Jose Manuel Barroso recently made similar claims.
In January, The Herald reported British diplomats were working behind the scenes to dampen down concern in Europe that Scotland's independence debate could trigger breakaway movements across the continent. Confidential communications showed the British ambassador in Madrid has been confronted with mounting fears that the 2014 poll will spark demands for similar polls in Catalonia and the Basque Country.
A Scottish Government spokesman said: "These comments have been overtaken by events because we now have a clear process by which Scotland can become an independent country, signed up to by both the Scottish and the UK Governments in the Edinburgh Agreement.
"As Mr Garcia-Margallo himself said earlier this year, this is an internal matter to be resolved within the UK – and we now have that clear agreement on the process."
The latest controversy came as Mr Salmond fought a rear-guard action aimed at preventing more defections from the SNP as the rebellion deepened over the change to the party's policy on Nato membership.
The First Minister launched an attempt to keep MSPs considering quitting in the fold following Friday's historic vote which saw the double resignation of Highland MSPs John Finnie and Jean Urquhart.
As the fallout continued from Deputy First Minister Nicola Sturgeon's admission that the Government waged a court battle at public expense for the sake of non-existent legal advice on EU membership, Mr Salmond addressed his MSPs' group meeting at Holyrood. As he attempted to lift the morale of his troops, it emerged Central Scotland MSP John Wilson may yet resign.
Efforts are being made to keep Mr Finnie and Ms Urquhart in favour with the party. They have not crossed the floor and are likely to vote with their former party.
On Tuesday, Mr Salmond had to issue a statement denying he was a liar, after it was revealed no advice had been sought from legal officers on EU membership in an independent Scotland. It came despite a TV interview in which he appeared to claim otherwise.
Mr Salmond was criticised by David Cameron at Prime Minister's Questions yesterday and by Labour MPs. Mr Cameron said the First Minister had got himself into a "truly baffling situation", which showed the case for independence fell apart under close scrutiny.
Mr Salmond also came under fire at a House of Lords committee hearing in Edinburgh on the economics of independence, and from Labour MEP Catherine Stihler, whose Freedom of Information request sparked the crisis. Former Nato general secretary Lord Robertson told the Scottish Review website the SNP's new Nato policy was "a disgraceful pretence" and "disingenuous commitment to the alliance".
Labour were furious at Holyrood at being refused a second emergency statement from the First Minister about the EU affair while the Tories demanded the Lord Advocate should be brought before the chamber.