The father of James Duke of Monmouth is usually assumed to be the future Charles II, who freely acknowledged his resonsibility. There exists a scientific proof, as published on p.36 of Beauclerk-Powell and Dewar’s Royal Bastards, through Y-chromosome tests comparing Monmouth’s male line descendants the Dukes of Buccleuch with the Dukes of Grafton, St. Albans and Richmond, from Charles’ other illegitimate sons.
Charles II was, of course, not unique in his Y-chromosome. In June-August 1648, when Monmouth was probably conceived in France, he was one of three brothers with a father still alive. Charles I was a prisoner in Carisbrooke Castle and Henry Duke of Gloucester was in England so they can be eliminated as Henry was also too young. Charles I was his father’s only surviving son and James VI/I had been his father’s only child.
From the attached document, you will observe that Henry Lord Darnley had one brother, who died without issue, and that his father (Matthew, Earl of Lennox) had two other sons but one was a childless Catholic Bishop. The other son was Jean Stuart, Seigneur d’Aubigny, whose French son Esme Duke of Lennox was known as James VI/I’s “favourite”. Esme’s male line grandsons all fought for the Royalist cause and three were killed between 1642-5.
There were two others, James and Ludovic, although they were more likely to have been in England than France in summer 1648. Together with the future Kings Charles II and James II, they share a common Y-chromosome with nobody except fourth or more distant cousins. Despite James II’s reputation for promiscuity, similar to that of Charles II in many ways, this more rigorous analysis tends to support the traditional view, for once.
The document also now shows the origin of the Stewarts and how Matthew of Lennox’s Y-chromosome should have matched that of James V, before his son married that King’s daughter: