For preservation reasons, the letter written just six hours before her execution on 8 February 1587 was only due to be on display for six hours.
Queues forming outside the National Library of Scotland where the letter is on display prompted an extension until 7pm.
The 430-year-old letter has not been on display in the capital since 2009.
Mary was one of the most fascinating and controversial figure in Scottish history.
She fled to England in 1567 after abdicating the throne in favour of her infant son James after a reign rocked by murder, war and scandal.
There she was imprisoned for the next 20 years by her cousin, Queen Elizabeth I.
Mary’s last letter was written to her brother-in-law King Henri III of France in the early hours of 8 February 1587, just six hours before her execution at Fotheringhay Castle.