Spain is standing in the way of Scotland's ambitions to become an independent nation within the European Union because of fears that it could spark the break-up of the Spanish state.

Spanish officials have registered concerns with counterparts in the United Kingdom over the Scottish government's independence blueprint, senior Whitehall sources confirmed yesterday.

Spain has indicated it could block an independent Scotland's accession to the European Union, sources said. It has already refused to recognise Kosovo's existence as an independent state. Madrid fears such moves will encourage separatist ambitions in Spanish regions, particularly Catalonia and the Basque region. Spain's refusal to recognise Kosovo has frustrated the former Serbian province's ambitions to enter the union.

The Catalan premier, Artur Mas, last week drew parallels with Scotland as he argued for a new financial deal with Madrid. "Spain refuses to speak publicly about Scotland at this stage," a senior Foreign Office source said yesterday. "But they have been making it clear for a number of years they are apprehensive about the prospect of Scotland becoming independent. The renewed debate about the referendum has started it all again."

A senior UK minister said: "We understand the Scottish view is they would wish to join the UN but they would not wish to join Nato. They might wish to join the EU, but we fully expect Spain to block it, fearing it might encourage the separatist spirit on their doorstep."

A Spanish veto would undermine claims an independent Scotland could immediately operate as a viable state. Although Alex Salmond insists Scotland would be able to join the EU following a "yes" vote in a referendum, experts maintain membership would not come automatically.

An official House of Commons briefing paper on the subject points out decisions on membership must be agreed unanimously by all EU member states. It added: "It is worth considering, if there is a continuing UK, it would have a vote on an independent Scotland's accession application, as would other member states with their own internal regional independence issues, such as Spain."

Professor Thomas Giegerich, an international law expert at Edinburgh University, said it would be "diplomatically difficult" for Scotland to join the EU after a "yes" vote, with other member states "reluctant" to anger the UK. However, a Scottish National Party spokesman said it was "preposterous" to suggest that Scotland could be excluded from the EU. He said: "Scotland has been an integral part of the EU for almost 40 years. An independent Scotland would be a succession state, not an accession state, and there is no provision for citizens of the EU to be expelled."

The implications an independence vote could have beyond UK borders emerged as the latest front in a war of attrition between London and Edinburgh. Mr Salmond will begin a counteroffensive this week with the launch of a consultation on an independence referendum. On Tuesday, the First Minister will be in London to promote the idea of a "new union of independent countries".