Restriction endonuclease fragment length polymorphisms (RFLPs) were found using the cDNA probe Hox-2.1 for the homeo box-2.1 gene in the mouse. Polymorphism was detected in restriction patterns generated by fragments from HindIII digestion. The great majority of laboratory strains of mice carries the Hox-2.1a allele. Only two laboratory strains carry the Hox-2.1b allele. Among strains of wild origin, the European subspecies (Mus m. domesticus, M. m. brevirostris, and M. m. musculus) and some Asian subspecies (M. m. castaneus) carry the Hox-2.1a allele. The subspecies from Far Eastern countries (M. m. molossinus, Chinese mice of wild origin, and M. m. yamashinai) carry the Hox-2.1b allele. Using the RFLP, the Hox-2.1 gene was mapped on chromosome 11. Three-point cross test data showed that the recombination frequency is 29.6% between the Hba and the Hox-2.1 genes and 23.5% between the Hox-2.1 and the Es-3 genes. The gene order of Hba-Hox-2.1-Es-3 has been confirmed.