A senior UK government minister has said it would "of course" refuse to give permission for a second independence referendum.
Speaking during a visit to Glasgow, Foreign Secretary Jeremy Hunt said the answer to any request for another vote would be "no".
First Minister Nicola Sturgeon said in January she would give an update on her plans for a referendum "in weeks".
But she has stressed that she will not hold a referendum without an agreement.
The UK and Scottish governments signed an agreement in October 2012 which allowed the Scottish Parliament to legislate for the independence referendum to be held two years later.
Ms Sturgeon told BBC Scotland on Thursday afternoon that she was "not open to the possibility" of another referendum being held without a similar agreement in place.
There have been calls from some within the independence movement for an unofficial referendum to be held, similar to the one in Catalonia in 2017, if the UK government's position does not change.
But Ms Sturgeon said: "My view is clear and always has been clear. The legal basis of any future independence referendum should be the same as the referendum in 2014, which is the transfer of power under a section 30 order.
"Of course the only reason we're talking about this is because of the anti-democratic stance of the Conservatives, who I think are running so scared of the will of the Scottish people on independence.
"They refuse to acknowledge the democratic mandate that the Scottish government has."
She was speaking after video footage emerged on the Broadcasting Scotland channel on Youtube of SNP deputy leader Keith Brown telling activists in Aberdeen last month that: "If we want to have a referendum, then we decide we're going to have a referendum".
The Scottish Conservatives claimed it proves the SNP is "planning for an illegal referendum" - but Mr Brown said his comments had been misinterpreted.
In a statement, he said: "My position is clear - the deeply undemocratic stance of the UK government in denying the mandate for indyref and refusing a Section 30 order should not prevent the Scottish government seeking one and planning on the basis of winning that case."
Several sources told the BBC last month that the UK government was preparing to reject any call from the Scottish government for the power to hold another referendum.
Mr Hunt confirmed this was the case as he was asked by journalists during a visit to Glasgow University whether Mrs May's response should be "yes or no".
The foreign secretary said: "The answer of course would be no for the very simple reason that we think the Scottish government should be focusing on the concerns of Scottish voters, which is not to have another very divisive independence referendum but to focus on an education system which used to be the envy of the world and standards are now falling, to focus on long waits in the NHS.
"That's what Scottish voters want the Scottish government to focus on and I am sure that that is what Theresa May will tell Nicola Sturgeon if she makes that request."
'Fog of Brexit'
Ms Sturgeon called for a new independence vote in the aftermath of the EU referendum, which saw 62% of Scottish voters back remain only for the UK as a whole to vote to leave.
However, the SNP leader subsequently "reset" her timetable after her party lost 21 seats in the snap general election of 2017.
Having previously said she must "wait for the fog of Brexit to clear" before settling on a new plan, Ms Sturgeon told MSPs on 17 January that she would outline her thoughts on the timing of a second independence referendum within "weeks" - even if Brexit was delayed.
The SNP say its 2016 Holyrood election manifesto gives them the right to hold another vote.
Ms Sturgeon's party won that election, with the manifesto including a commitment that another referendum could be held if there was a significant change in circumstances from 2014 - such as Scotland being taken out of the European Union against the wishes of voters north of the border.