Long thought to belong to Henri, assassinated at the age of 57 by a Catholic fanatic in 1610, the head changed hands several times over the next two centuries, bought and sold at auction or kept in secretive private collections.
Scientists in 2010 said they found proof that the head was indeed Henri's, citing physical features that matched 16th century portraits of the king, as well as radiocarbon dating, 3D scanning and X-rays.
The 2010 study, however, found no DNA and its findings have been contested by some.
With the new evidence, "it is about 250 times more likely that the (owners of the) head and the blood are paternally related, than unrelated," co-author Carles Lalueza Fox of the Institut de Biologia Evolutiva in Barcelona said by email.
Taken together with all the physical and forensic evidence, historical records and folklore, it would be "extremely surprising" if the remains did not belong to the two assassinated monarchs, he added.
"One can say that there is absolutely no doubt anymore," about the authenticity of the mummified head, added Charlier.