Her second husband (Lord Darnley) murdered her secretary

When Mary was in mourning over Francis, she briefly encountered her English-born half-cousin Henry Stuart, Lord Darnley. The Earl and Countess of Lennox, Darnley's parents, were both Scottish aristocrats and English landowners. They sent him to France to express their grief while also hoping for a possible match between their son and Mary. Mary and Darnley were both the grandchildren of Margaret Tudor, Henry VIII of England's sister, and patrilineal ancestors of the High Stewards of Scotland.

Darnley soon became haughty. Not satisfied with his position as king consort, he sought the Crown Matrimonial, which would have given him a co-sovereign of Scotland with the right to retain the Scottish throne if he outlived his wife. Mary declined his proposal, and their marriage became tense, despite the fact that they had pregnant by October 1565. He envied her bond with her Catholic private secretary, David Rizzio, who was thought to be her child's father. Darnley had formed a secret plot with Protestant lords, including the nobility who had risen against Mary in the Chaseabout Raid, by March 1566. On March 9, her second husband murdered her secretary. A gang of conspirators led by Darnley assassinated Rizzio in front of the pregnant Mary at a dinner party at Holyrood Palace. Darnley switched sides during the next two days, and Mary received Moray at Holyrood. Darnley and Mary escaped from the palace on the night of March 11-12. They took a safety in Dunbar Castle before returning to Edinburgh on March 18. Lords Moray, Argyll, and Glencairn were reinstated to the council.