The UK Government has warned that the cost of international travel could increase for Scottish passport holders.
It claimed an independent state would not automatically inherit the waiver agreements which entitle UK passport holders to travel to 173 countries without paying for visas.
The Scotland Office said the situation would have implications for travellers and the economy – and could dissuade tourists from visiting the country.
Civil servants said the bilateral agreements currently in place were between the UK and other nations, and if an independent Scotland wanted to have similar agreements they would have to be negotiated.
A report titled Borders and Citizenship published yesterday said: “It cannot be assumed that an independent Scottish state would inherit the visa waiver agreements negotiated by the UK.”
The report said Scottish passport holders would have to pay £106.35 for a visa to visit America if a deal was not struck between Edinburgh and Washington.
“In 2009 there were 459,656 passenger journeys from Scotland’s main airports to the USA,” it said.
“This would equate to 229,828 US visas (non- immigrant) if these were return trips made on a Scottish passport and there was no visa waiver agreement.
“At a cost of £106.35 each this would mean the loss to the Scottish economy and transfer to the USA of £24.4million a year.”
Scottish Secretary Alistair Carmichael said: “As part of the UK we have the benefit of bilateral arrangements that have been constructed over decades and centuries.
“That is yet one other aspect of international obligations that remain with the continuing UK Government from which an independent Scotland would have to walk away and cut their own series of new bilateral deals.
“Would an independent Scotland be able to get deals with all those countries that we currently have?
“Maybe yes, maybe no, it would all have to be negotiated.”
Scottish Conservative constitution spokeswoman Annabel Goldie said: “This is yet another uncertainty which can be added to the huge list of uncertainties that come with separation.”