Counting will be carried out through the night, with individual results announced for each of Scotland’s 32 local authority areas.
The final national result is expected after 06:30 BST on September 19.
A YouGov on-the-day poll published shortly after polls closed suggested “No” was on 54% and “Yes” on 46%.
The survey questioned 1,828 people after they voted, together with the postal votes of 800 people, although is not a traditional exit poll.
Turnout is widely predicted to top the 83.9% recorded in the 1950 general election – the highest in the UK since the introduction of universal suffrage in 1918.
Blair McDougall, director of the pro-Union Better Together campaign, said he believed a “No” vote would be revealed over the course of the night.
A “Yes” vote in the ballot would end the 307-year-old union between Scotland and the rest of the UK.
A record turnout is anticipated, with 4,283,392 people – 97% of the electorate – having registered to vote.
As soon as Scotland’s 2,608 polling places closed, work began to transport hundreds of ballot boxes to counting centers in each of Scotland’s 32 local authorities.
The bulk of the local results are expected to come between 03:00 and 05:00 BST on September 19.
These will include votes cast from the 789,024 postal vote applications, which was the largest volume of registration for postal votes ever in Scotland.
Once the results from all the local authority areas are known, chief counting officer Mary Pitcaithly will declare the result of the referendum at the Royal Highland Centre outside Edinburgh.
However, running totals – which can be made from the first declaration onwards – may indicate a result earlier in the morning.
Opinion polls released before the vote suggested the referendum was too close to call, although most had the “No” campaign – which backs staying in the UK – holding a slight lead.
For the first time, 16 and 17-year-olds all across Scotland were able vote.
Many councils have reported busy polling stations were busy throughout the day, with some seeing queues both ahead of the polls opening and throughout the morning.
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