Senior EU officials have for the first time hinted that an independent Scotland would need to make its own application for EU membership, reports The Daily Telegraph.
Speaking on the matter, EU Commission President Manuel Barroso revealed that new nation states, such as a devolved Scotland or Catalonia in Spain, would need to apply for EU membership themselves under international law.
"To join the EU, yes, we have a procedure and it is a procedure in international law," Mr Barroso told the BBC.
"A new state, if it wants to join the EU, has to apply to become a member of the EU like any state," he added.
North of the border the ruling SNP party is negotiating plans to hold a referendum on Scottish devolution and had hoped that Scotland's existing EU membership as part of the UK would suffice.
However, Olivier Bailly, a spokesman for the European Commission, said that any country formed from an existing member state would be treated as an 'accession state'.
EU membership is valuable and can take many years to achieve. Turkey first applied to join the EU in 1987 but it took 12 years for it to be recognized as a candidate for full membership. Twenty-five years after first applying, Turkey remains outside the EU.
Whilst it is accepted that Scotland poses far fewer political and military issues than a power such as Turkey, there can be little doubt that failure to automatically join the EU will cost Scotland millions in lost trade and could have a disastrous effect on the Scottish economy.
There are two different steps to acquiring EU membership. First a newly formed country must undergo a process known as secession. Then the country must apply for accession in accordance with the various EU treaties.
The claims have angered the SNP, which believes that there is no existing process for newly formed countries to have to secede to the EU. The SNP believe that a devolved Scotland would continue membership of the EU.
Keith Brown is the Transport Minister for Scotland.
"It is simply our position, and it always has been, that Scotland will accede to the EU simply because we take on the same obligations and treaties that we currently have," he told the BBC.
SNP leader Alex Salmond believes that Scotland will have to negotiate with the EU, but he firmly believes that a devolved Scotland would keep its existing membership as part of the UK.
Anti-devolution group Better Together has jumped on the words of the EU Commission as evidence that devolution would be bad for Scotland.
"We now have it straight from the horse's mouth. The SNP's assertion that we would be granted automatic entry has proven to be, like so many of their arguments, a baseless assertion."
One major point for discussion in a devolution scenario is that of currency. At present Scotland uses the British pound, albeit with its own notes. Experts believe that a devolved Scotland would be forced either to issue its own currency or join the Euro.
At the moment, the UK Government has its own commission looking into the legal ramifications of devolution.
A Foreign Office spokesman said: "The UK Government is undertaking its own detailed research into the benefits of Scotland's place within the UK, including membership of international organisations."