Nicola Sturgeon's plan to split from the rest of the UK and maintain Scotland's "relationship with Europe" took a blow on Tuesday (14 March).
A source close to the EU Commission, the executive arm of the economic and political bloc, confirmed to IBTimes UK that the so called "Barroso doctrine" still applies.
The policy is named after former EU Commission President José Manuel Barroso, who in 2012 told the BBC that an independent Scotland would have to negotiate its membership of the EU.
"For European Union purposes, from a legal point of view, it is certainly a new state," the Portuguese politician explained.
"If a country becomes independent it is a new state and has to negotiate with the EU."
The doctrine has significant implications for Sturgeon and the SNP since re-joining the economic and political bloc could require Scotland adopting the Euro currency, according to the 1992 Maastricht Treaty.
But the nation could fail to meet the five "euro convergence criteria" or the "Maastricht criteria" to adopt the single currency.
Spain may also scupper an independent Scotland's talks with the EU in a bid to tackle calls for Catalonian independence. A referendum is to be held in the north-eastern region of Spain in September 2017.
Sturgeon will seek Holyrood's approval for a second Scottish referendum next week and plans to hold the ballot between Autumn 2018 and Spring 2019.
Scots voted 55% against splitting from the rest of the UK in 2014. But 62% of Scottish voters backed a Remain vote at the EU referendum in June, when the UK voted 52% to 48% to split from the economic and political bloc.
Theresa May has the constitutional authority to hold another referendum as prime minister for the UK.
But David Cameron gave the SNP a precedent with his 2012 agreement with former First Minister of Scotland Alex Salmond.
"The UK government was clear in 2014 that an independence referendum should, in their words, be made in Scotland by the people of Scotland," Sturgeon said. "That is a principle that should be respected today. The detailed arrangements of a referendum, including its timing, must be for the Scottish Parliament to decide."