Speaking in the House of Commons, the Cabinet Office minister said given only six years had passed since the 2014 referendum it was too soon to hold another one.
“In 2014 Nicola Sturgeon and other leading Scottish nationalists made the point that that referendum was for a generation,” Mr Gove said in response to a question from the SNP’s Kenny MacAskill.
“Just six years later, I do not believe a generation has passed.”
The SNP government in Edinburgh has insisted the Scottish Parliament should be given the powers to decide itself whether to hold another plebiscite, which is currently something only Westminster can approve.
Scottish Independence: For and against
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Boris Johnson has resisted such calls, although polling shows two-thirds of Scots agree there should be a second referendum should Nicola Sturgeon’s party win a majority in the upcoming Holyrood election next year.
Mr MacAskill claimed in the Commons even senior Tories were now coming round to the “inevitability” of another vote on independence soon.
Earlier this week the former prime minister John Major urged Mr Johnson to call two referendums – one on the principle of independence and a second to approve the actual negotiated outcome – as a way of drawing the sting of the growing movement for secession.
“Scots have learned, as the Secretary of State will know, from the trickery of 1979 when even the dead were counted against,” he warned Mr Gove.
The East Lothian MP was referring to a previous devolution referendum which was rejected by Westminster despite recording a narrow victory for the pro-devolution camp because of rules which stated more than 40 per cent of the total electorate had to vote in favour.
The SNP has long claimed out-of-date voter rolls, which may have included electors who had died before the plebiscite was even held, meant this stipulation could never have been reached.
“Does the Secretary of State not then realise that the people of Scotland will not accept political chicanery on the number or nature of the question to be asked?” Mr MacAskill also said.
“He makes a very, very important point,” Mr Gove replied. “It’s absolutely vital we have confidence in the integrity of our democratic institutions and that’s why the Electoral Commission and other bodies play such an important role.
“But of course it’s also important people can have confidence in the promises made by politicians.
“And it was the case in 2014 that Nicola Sturgeon and other leading Scottish nationalists made the point that that referendum was for a generation.”
Earlier this month, the Scottish secretary Alister Jack suggested a generation was between 25 and 40 years, implying the Conservative government would not support a second vote until 2039 at the earliest.