Jeremy Corbyn has said he would not allow a Scottish independence referendum "in the formative years" of a Labour government.
But the Labour leader, on a visit to Scotland, said the party would not block a new poll at a future date if there was a "fresh mandate"
Mr Corbyn insisted independence was "not the answer" to Boris Johnson.
His comments follow a row within Labour over whether a Labour UK government should seek to block a new referendum.
The Labour leader was starting a three-day visit to Scotland in Dunfermline, speaking to local people about the fire at Woodmill High School.
Mr Corbyn said: "Boris Johnson's pursuit of a no-deal Brexit will be a disaster for the whole country. He is putting at risk Scottish jobs in manufacturing, food processing and service industries. His intention to suspend Parliament shows he is also a threat to our democracy.
"The best way to defeat Johnson is sticking together, electing a Labour government and allowing it to get on with sorting the Tory Brexit nightmare and introducing policies that will see transformative investment in Scotland's people, communities and public services.
"Scottish independence is not the answer to Johnson. Independence will only further prolong and intensify austerity and create more instability and chaos."
Clarifying Labour's likely response if the Scottish government formally requested a new vote on independence, he indicated it would be a question of timing.
"In the formative years of a Labour government we wouldn't agree to another independence referendum because we will be fully focused on these central priorities," he said.
"However, if at some future point there was a legitimate and fresh mandate, we wouldn't block it."
Scottish Labour has been opposed to another ballot on independence.
But UK shadow chancellor John McDonnell re-opened debate on the issue during a visit to Edinburgh, saying the party would not stand in the way of a second referendum.
Scottish leader Richard Leonard said he was in agreement with Mr Corbyn on the timing of a possible second ballot.
"Jeremy and I have agreed that, during the formative years of an incoming Labour government, we would not sanction a Section 30 order to allow a further referendum on Scottish independence to take place," he said.
Mr Leonard added: "It would also only be acceptable to a Labour government to allow a second referendum to proceed if it could be demonstrated that there was a fresh mandate for such a vote to be held. This would require a democratic mandate from the people of Scotland, which clearly signals the majority of people are in favour of a new vote.
"I do not believe that the conditions exist for such a move today, nor will they for the foreseeable future."
When Mr Corbyn spoke on the issue previously, First Minister Nicola Sturgeon welcomed Mr Corbyn's refusal to block a referendum.
She said: "It's perfectly legitimate to oppose independence, as he does, it's perfectly legitimate to argue against there being a second referendum.
"What's not legitimate is for Westminster to block a mandate as now exists for a second referendum, and to seek to block majority opinion in the Scottish Parliament."