Charities, such as the British Heart Foundation, Cancer Research UK and Diabetes UK have major presences north and south of the Border.
Labour MSP Richard Baker, who is a former press and policy officer for Help the Aged, said fundraising by some charities in England subsidises their work in Scotland.
Mr Baker warned that the creation of an independent Scotland would lead to different tax regimes and regulations north and south of the Border for charities that operate across the UK.
His intervention came after the launch of the Third Sector Yes campaign – a group of pro-independence supporters from charitable and voluntary sectors.
The Labour MSP suggested that some charities may have to consider breaking up their organisations, which he warned could lead to a potential loss of revenue for good causes.
Mr Baker said: “Some years ago, I worked for a UK charity and there’s no doubt that fundraising south of the Border helps support lots of charity services in Scotland.”
The Labour MSP, a director of the anti-independence Better Together campaign, went on to say that a Yes vote in next year’s referendum could raise “very serious questions” about how major charities are organised in Scotland.
He said: “Clearly, if you have a UK charity that is geared up to work across a single state, it raises huge questions if there are then suddenly two states.”
Senior officials at two leading UK charities insisted they would maintain links with an independent Scotland, but would not say whether they would need to launch a separate Scottish organisation in the event of a Yes vote.
Simon Gillespie, chief executive of the British Heart Foundation, said: “We are committed to working across the four nations of the UK. Whatever happens on the political landscape, we will continue to work with the people of Scotland.”
Diabetes UK chief executive Barbara Young said: “Our response to the outcome of the referendum will be driven by the needs of people with diabetes living in Scotland.”
Gregor McNie, Cancer Research UK’s public affairs manager, said: “We will continue to work closely with the Scottish government.”
SNP MSP John Wilson insisted the work of UK-based charities would continue in an independent Scotland.
Mr Wilson said: “The work of what would become cross-border charitable organisations would be unaffected by a decision on independence.”
A spokesman for the Third Sector Yes said charities would benefit from full economic powers for the Scottish Parliament under independence.
The spokesman said: “Many charities already have successful cross-border arrangements and after a Yes vote these would continue.
“The big advantage of a Yes vote is that the Scottish Parliament would control both charity law and all tax matters, whereas at present only the former is devolved.”