The intraspecific variations of Entomophthora muscae s. str. associated with particular host species, Musca domestica and Delia radicum, sampled from different localities and different years in Denmark and the variation of E. muscae s. str. originating from different host taxa were investigated. The isolates were compared both by primary spore morphology and by three molecular methods: random amplified polymorphic DNA, universal primed PCR, and PCR-restriction fragment length polymorphism. Analyses of the different molecular data showed the same overall picture and separated E. muscae s. str. into two main groups with all the M. domestica isolates in one group and isolates from D. radicum, Coenosia tigrina, and Pegoplata infirma in the second group. E. muscae s. str. isolates from M. domestica also differ significantly from the rest of the E. muscae s. str. isolates with regard to the morphology of the primary conidia, which were bigger and contained significantly more nuclei per conidium. Several different E. muscae s. str. genotypes were documented and each type was restricted to a single host species, indicating a very high degree of host specificity at or below the level of the subfamily.