The “royal disease,” a blood disorder transmitted from Queen Victoria to European royal families, is a striking example of X-linked recessive inheritance. Although the disease is widely recognized to be a form of the blood clotting disorder hemophilia, its molecular basis has never been identified, and the royal disease is now likely extinct. We identified the likely disease-causing mutation by applying genomic methodologies (multiplex target amplification and massively parallel sequencing) to historical specimens from the Romanov branch of the royal family. The mutation occurs in F9, a gene on the X chromosome that encodes blood coagulation factor IX, and is predicted to alter RNA splicing and to lead to production of a truncated form of factor IX. Thus, the royal disease is the severe form of hemophilia, also known as hemophilia B or Christmas disease.