Liz Truss has insisted she will not allow a second referendum on independence to take place if she becomes the next prime minister.
The favourite to replace Boris Johnson as Tory leader claimed she would "do what is necessary and right to defend our Union" in comments which are likely to infuriate Nationalists.
Nicola Sturgeon has repeatedly said she has a clear mandate to hold another referendum following the SNP's victory at the Holyrood elections in May last year.
The First Minister announced to MSPs last month her government was planning to hold an IndyRef2 on October 19, 2023 - but only if the UK Supreme Court agreed such a move was legal.
Johnson has insisted "now is not the time" - but the comments from Truss show she is willing to take a stronger position.
"Scottish Nationalists accepted that their referendum was a once-in-a-generation opportunity, and I will hold them to that," she told the Sunday Telegraph.
"I will work to strengthen our whole Union. As prime minister, I will do what is necessary and right to defend our Union, just as I have already done on the Northern Ireland Protocol."
Truss continued: "The SNP lost the 2014 referendum and Nicola Sturgeon is now leading a campaign of deception to steamroller the UK and break up the Union.
"But I am completely clear that there will be no second Scottish independence referendum on my watch.
"The Scottish Independence Referendum Bill isn't legal and it will be invalidated if passed by the Scottish Parliament. When Westminster devolved power to Scotland, it did not include the ability to hold valid referenda to break up the Union.
"Any Scotland independence referendum would need to be authorised by the Westminster Parliament. If I become prime minister, I would not grant that authority."
The First Minister has previously explained her preference for staging an IndyRef2 would be to follow the example of the 2014 vote.
That saw the UK Government lend Holyrood the powers required via a section 30 order.
But Johnson has made clear he has no intention of agreeing such a deal while he remains in Downing Street.
Sturgeon has said her party will use the next general election as a proxy referendum if her plans for a vote next October are thwarted.
Responding to the comments by Truss, SNP depute leader Keith Brown said: "It is crystal clear that the people of Scotland and Scottish democracy mean nothing to either of the contenders for next Tory Prime Minister.
"But people across Scotland will not stand for yet another Tory Prime Minister we didn't vote for denying the cast iron mandate to hold an independence referendum of a government and parliament we overwhelmingly voted for.
"With each day that passes, both Tory candidates are showing how out of touch they are with people across Scotland as they prioritise trying to outdo each other on Brexit, ducking their duties to support struggling households and standing in the way of Scotland's right to choose its future - Scotland deserves so much better.
"Scots voted for the chance to choose a better future in an independence referendum and - whoever becomes the next leader of the Tories - that future as a wealthier, fairer and more equal independent country is more vital than ever."
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