The Pontiff, in an interview with a Barcelona newspaper about Catalonia's conflict with Spain, said "all division worried him".
But he also cited other political independence movements emerging in Europe, such as in Scotland and northern Italy's Padania, adding that they needed further study.
The Pope contrasted the emancipation of nations in his native Latin America with modern secessionist movements.
Asked specifically about the conflict between Spain and Catalonia, he said: "All division worries me."
Speaking to La Vanguardia, he added: "There is independence for emancipation and there is independence for secession.
"We think of the old Yugoslavia, where there are peoples and cultures so diverse that they are completely unconnected.
"The Yugoslav case is very clear but I ask myself if things are quite as clear in the other communities that have been together up to now.
"They should be studied on a case-by-case basis. Scotland, Padania, Catalonia. There will be cases that are right and ones that are not."
Campaigners on both sides of the indyref have welcomed the Pope's comments.
A spokesman for the pro-independence Yes Scotland campaign said: "As His Holiness says, these matters should be looked at on a case-by-case basis."
"Scotland's national status has always been recognised by the Catholic Church - indeed, Scotland has been a filia specialis, or favourite daughter, of the Papacy.
"The aspiration to independence is about making Scotland a fairer country - where wealth is shared more equally among all the people who live here, and we banish nuclear weapons once and for all."
Commenting on behalf of the pro-UK Better Together campaign, Ms McGuire, the MP for Stirling, said: "This is an interesting and welcome intervention from Pope Francis.
"The Pope is right to warn about the impact of division on society and to draw the distinction between independence for emancipation and that for secession.
"We live in a large interdependent world and the best way to secure our future is to work together as part of something bigger.
"Coming on the back of the interventions by President Obama, Hillary Clinton and JK Rowling, the comments from Pope Francis highlight just how important the decision we have to make in September is."
Scottish Liberal Democrat leader Willie Rennie said: "This thoughtful contribution from the Pope rightly urges people to be careful when considering independence.
"Drawing a distinction between different kinds of independence will help people here consider the motivation for this change.
"The Pope is clearly anxious that any change is done for good reasons and is concerned about dismembering of nations.
"That the Pope speaks on this shows how serious this referendum is. That in itself should be pause for thought."