Under the present system, students from other European Union states are entitled to free university education, while people from England, Wales and Northern Ireland can be charged fees of up to £9000 a year.
If Scotland became a new European state, people from the rest of the UK would fall under the same EU exemption – leaving a shortfall of cash for universities.
Ministers had originally suggested the funding gap would be £155 million, but new figures from the Scottish Parliament suggest it could be as much as £263m by 2014/15.
Liz Smith, education spokeswoman for the Scottish Conservative Party, said: "At a time when the overall competence of the Scottish Government has been seriously undermined, yet more questions have arisen about the size of the funding gap and the full cost of higher education.
"The Scottish Parliament Information Centre has estimated the Scottish Government has already underestimated the funding gap by more than £100m."
However, a Scottish Government spokesman said the claim was based on out-of-date figures and did not reflect the investment put into Scottish universities in the most recent spending reviews.
"Funding arrangements for EU students, which will include students from the rest of the UK post-Independence, are currently being considered and at this point predictions of what those arrangements might be or what number of students from the rest of the UK might apply to Scottish universities, are purely speculative," he said.
In a separate development, the Scottish Liberal Democrats are to reaffirm their support for free university tuition for Scottish students. NUS Scotland welcomed the move.