Changes to the regulatory environment or the availability of public funding as a result of the referendum, or from next year's UK general election, "could affect the group's prospects", Stagecoach said in its preliminary results.
The referendum is included in the "principle risks and uncertainties" facing the company, a list which also includes catastrophic events, terrorism, economic environment, rail cost base, breach of franchise, pension scheme funding, insurance claims, disease, IT systems failures and potential malicious attacks and Treasury changes in fuel prices, interest rates and exchange rates.
The report said, under the heading Regulatory changes and availability of public funding: "There is a risk that changes to the regulatory environment or changes to the availability of public funding could affect the group's prospects. Such changes may arise as a result of the outcomes of the September 2014 referendum on Scottish independence and/or the 2015 UK general election."
But Sir Brian, the group's chairman, said Stagecoach has made a "satisfactory" start to 2014/15 with a "positive" outlook for the year ahead.
Sir Brian said: "The group is in a strong financial position, with investment grade credit ratings, and I believe the prospects for our customers, employees and our shareholders are positive."
He has pledged to gift up to £1 million to the SNP by matching pound-for-pound every donation received during the year of the independence referendum.
Sir Brian has also donated £100,000 to Yes Scotland-affiliated group Christians For Yes.
He donated £500,000 to fund the party's 2007 election campaign and match-funded a further £500,000 ahead of the SNP's landslide 2011 win.
Scottish Labour MP and Shadow Business Minister Ian Murray said: "When even Brian Souter, who has bankrolled the nationalists for years, thinks separation is a risk then you know Alex Salmond has a problem. This intervention drives a coach and horses through the nationalists' case for separation.
"Being part of the UK single market works well for firms like Stagecoach. Scottish employers have access to a market of more than 63 million people across the UK, rather than just five million people in Scotland. Where is the sense in putting up barriers between Scottish firms and their customers based elsewhere in the UK?
"As part of the UK we can have the best of both worlds for Scotland. We can have our strong Scottish Parliament, with the guarantee of more powers for Scotland, backed up by the strength, security and stability of being part of the larger UK. Alex Salmond wants us to put that at risk on the strength of nothing more than his empty promises."
Conservative deputy leader Jackson Carlaw said: "What a hoot that Stagecoach is warning about independence even while its chairman remains an avid fan.
"Surely it's pretty ironic for Brian Souter to wager an each-way bet, mouthing off and bankrolling Yes Scotland on the one hand, despite it seems, his views as the public face of Stagecoach being far from representative of the very company he has built up from scratch.
"Clearly, Scotland and Stagecoach realise that independence is a one-way ticket to a poorer nation."
Business for Scotland member David Cairns, executive chairman of IT firm Prismtech, said: "This is a nonsensical interpretation by the No campaign.
"Brian Souter is on the record as a business leader who will vote Yes in September so that we can build a more prosperous economy and fairer society, so we should be grateful to the No campaign for drawing attention to his strong pro-independence views.
"The report discusses the UK general election in exactly the same context as the referendum and is therefore not about an independent Scotland - just as it doesn't specify any particular outcome of the 2015 general election."
A Stagecoach Group spokesman said: "Consistent with other companies, we ensure our investors are aware of relevant matters affecting our business, both risks and opportunities.
"This section of our preliminary results makes the point that our business operates in an environment which can be subject to political change.
"It highlights the September 2014 referendum on Scottish independence and the 2015 UK general election as forthcoming events which could result in change.
"It is not a comment on the merits of Scottish independence, which we have consistently stated is a matter for the people of Scotland to decide.
"Stagecoach Group remains neutral on the issue, as it has always been."
Liberal Democrat leader Willie Rennie said: "When it comes down to the nitty-gritty of his business even Brian Souter recognises the risks of independence.
"Alex Salmond is so passionate about his independence plans that he refuses to do the same rational calculation of the facts. Brian Souter knows that he can't throw his shareholders under the bus.
"The questions on Alex Salmond's independence plans are stacking up day by day.
"With concerns now coming from their strongest supporters, the nationalists should give people the facts they deserve on issues like set up costs, EU membership and currency."