Hybrid sterility can prevent gene flow between diverging subpopulations and hence might contribute to speciation. The hybrid sterility 1 (Hst1) gene was originally described in male progeny obtained from a cross between laboratory inbred mouse strains C57BL/10 and C3H, and wild Mus musculus musculus, sampled at localities in Prague, Czech Republic. This study asked whether the presence of sterility associated with the Hst1 gene is limited to one local population or is extended over geographically distant regions. We studied the progeny derived from a wild population of M. m. musculus sampled in Studenec, Czech Republic, 160 km south-east from Prague, crossed reciprocally to C57BL/10 mice. Spermatogenesis was examined in 251 hybrid males; among them 109 males (43.4%) were sterile and 142 (56.6%) were fertile. Sterile males had significantly lower testis mass and lower epididymis mass compared with fertile males. The size of the reproductive organs was dependent on cross reciprocity within the classes of sterile and fertile males. Although our phenotype data resemble those presented in the original description of Hst1, molecular analysis revealed incomplete segregation of sterility and fertility in male progeny and markers from the Hst1 region in some families. Therefore, there are probably additional genes affecting hybrid sterility that are polymorphic in wild M. m. musculus. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 663–674.