We developed new Entomophthora-specific primers for nested-PCR of the ITS II region to be used on in vivo material and combined it with RFLP. Resting spores from Scathophaga stercoraria (3 specimens), Delia radicum (9 specimens), Botanophila fugax (1 specimen), and two syrphid host species, Platycheirus peltatus and Melanostoma mellinum (one specimen of each) were characterized genetically after analysis of RFLP-profiles of the PCR-products. The genetic characterization of the resting spore isolates was compared with twenty isolates of known primary conidial morphology (in vitro and in vivo) from the E. muscae species complex. The analysis allowed for the first time a separation of resting spore isolates into the species level, which is not possible only using morphological characters (diameter). Isolates originating from different specimens of the same host taxa appeared to be strongly clonal even they were sampled at different localities in different years. Isolates morphologically belonging to E. muscae s. str. (e.g., including E. scatophagae) could be separated genetically further into sub-groups entirely depending on the host taxa; each fungal genotype, either present at the conidial stage or at the resting spore stage, is correlated with one host species. Furthermore, E. muscae s. str. originating from D. radicum proved to be much more closely related to E. scatophagae than to E. muscae s. str. originating from M. domestica. None of the resting spore isolates could be assigned to E. schizophorae. The nested-PCR approach accompanied by RFLP proved its usefulness for identification of resting spores and for more detailed studies clarifying host-pathogen specificity and interactions. It seems that different members of the E. muscae species complex are able to complete their life cycle in only one host species and, further, that each pathogen-host system is independent.