It seemed that Darnley had a close friend named David Rizzio. Rizzio was appointed secretary to Mary and they became so close that he was suspected of being her lover. At eight in the evening of 9th March 1566, Mary, Rizzio and some guests were taking supper in a room nesr her bedchamber in Holyroodhouse Palace in Edinburgh, when a group of men burst in. This gang, which included Darnley, was led by Lord Ruthven, a Protestant Scot more concerned with Marys' Catholicism than her morals. Rizzio hid behind Mary (then pregnant with James VI of Scotland, later James I of England). Rizzio was dragged out, taken to another room and stabbed 57 times before his corpse was stripped and thrown down the stairs. After that ,Mary and Darnley lived separately.

In 1567, Darnley took lodgings in the Old Provost House in the ground of Kirk o'Fields, a large church then on the outskirts of Edinburgh. Mary would visit him there occasionally. Early in the morning of 10th February 1567, the Old Provost House was blown up by a gunpowder charge so large that the entire building was demolished. The bodies of Darnley and a servent were found, partially-clothed, in the nearby orchard. Neither had any injuries from the explosion, but those who examined Darnleys' body believed he had been strangled with a ligature.

One of the chief suspects was a Lord Bothwell, but no evidence could be found to implicate him. Mary married him three months later.