The locations of five random mouse genomic DNA markers and five cloned genes, including the genes for clotting factors VIII and IX (Cf-8 and Cf-9), Duchenne muscular dystrophy (Dmd), phosphoglycerate kinase-1 (Pgk-1), and alpha-galactosidase (Ags), on the mouse X chromosome were determined by in situ hybridization. The five random DNA markers provide new genetic loci with useful restriction fragment length polymorphisms between mouse strains and species, including one locus close to the centromeric region of the mouse X chromosome. The physical map and the recombination map of these loci on the X chromosome were compared. There was good agreement in the order of loci. Relative distances between loci were consistent along the X chromosome, with the exception of the telomeric end of the long arm, where the recombination fraction observed between loci closely associated on the physical map was higher than that between similarly spaced markers located in the proximal region of the X chromosome. These results are discussed in comparison to the human X-chromosome map.