The head was lost after revolutionaries ransacked the royal chapel at Saint Denis, near Paris, in 1793.
A head, presumed to be that of Henri IV, has passed between private collectors since then.
A team of scientists used the latest forensic techniques to identify features seen in portraits of the king.
A lesion near his nose, a pierced ear and a healed facial wound – from a previous assassination attempt – were among the marks that identified the head.
The methods used to embalm the head also matched techniques in use at the time of his death, said the scientists in a report published by the British Medical Journal (BMJ).