Darnley was the second son of Matthew Stewart, 4th Earl of Lennox, and his wife, Lady Margaret Douglas. He was their oldest surviving son. Darnley's maternal grandparents were Archibald Douglas, 6th Earl of Angus, and Margaret Tudor, widow of James IV of Scotland. It is the common belief that Henry Stewart was born on 7 December, but this is disputed. He was a first cousin and the second husband of Mary, Queen of Scots, and was the father of her son James VI of Scotland, who succeeded Elizabeth I of England as James I.