Mary I was born in Linlithgow palace in 1542 and became queen of Scotland a week later when her father King James V died. She was the first undisputed reigning queen in these islands, and also the first called Mary. The English attempted to force a marriage treaty between Mary and their Prince Edward, hoping to control Scotland, but the Scots made a treaty with Henry II of France betrothing Mary to Henry's son Francis, and she was sent to live in France in 1548. While Queen Mary was absent, Scotland was ruled in her name first by her cousin James Hamilton earl of Arran, and then by Mary's French mother Mary of Guise-Lorraine. Already queen of Scots in her own right, Mary also became queen of France in 1559 with her husband Francis, and they laid claim to the crown of England on the grounds that Mary was the legitimate Catholic heir. But in 1560 Francis died. That same year Scottish religious reformers seized power in Scotland with the backing of the English who wished to engineer regime change and end French influence. A parliament was held in Edinburgh, which was technically illegal, and declared Scotland to be a Protestant country.

In 1561 Queen Mary returned to Scotland after nearly 13 years absence. Mary's natural half-brother Lord James Stewart was at the head of the Protestant party and proved a formidable cause of instability in the kingdom which was not helped by Mary's lack of political judgement. In 1565 she married her Catholic cousin Henry Stewart, to which James Stewart and other nobles objected, revolted and were exiled for a time. They returned and murdered the queen's Italian secretary David Riccio in a dramatic night at Holyrood Palace, in which Mary's husband Henry was involved. Then Henry was murdered in turn by his fellow conspirators. When Queen Mary married James Hepburn earl of Bothwell – the man who was now chiefly rumoured as the murderer of King Henry – there was a great scandal which enabled Mary's enemies to rise against her. In 1567 Bothwell was forced to flee and Mary surrendered. She was made to abdicate in favour of her infant son, who was now crowned James VI, and she was imprisoned on Lochleven, while Lord James ruled Scotland as regent. In 1568 Mary made a dramatic escape, raised another army, and, suffering defeat at the battle of Langside, fled across the border to England hoping to secure aid to regain her throne.

Queen Elizabeth of England, whom Catholics regarded as an illegitimate usurper, was alarmed by the presence of Mary in her kingdom. Lord James, and the regents who followed him, had deposed Mary and did not want her back in Scotland, so she became a focus for Catholic discontent while she remained in exile in England. For 19 years Elizabeth was forced to detain Mary under house arrest in different places, while various plots were hatched, until finally, Mary was brought to a show trial, condemned, and then beheaded in 1587. The legality of Mary's trial and execution, as a foreign, deposed monarch, was extremely dubious, and Elizabeth was reluctant to consent to the death of a fellow anointed queen for fear of setting a precedent. In the end, though, Mary had become too much of a danger to be ignored and it was either her or Elizabeth.