Three nematode-endoparasitic, Hirsutella rhossiliensis (18 isolates), Hirsutella minnesotensis (8 isolates) and Hirsutella vermicola (3 isolates) were studied for their variability in morphology, nematode parasitism, and DNA sequences of internal transcribed spacer (ITS) of ribosomal DNA (rDNA) and mitogen-activated protein kinase (MAPK) gene fragments. There were differences in length and width of conidiogenous cells and conidia among the three Hirsutella species and among isolates within species of H. rhossiliensis and H. minnesotensis. Most isolates of H. rhossiliensis and H. minnesotensis parasitized higher percentages of the cyst nematodes, Heterodera glycines and Heterodera avenae, than the four non-cyst nematodes, Meloidogyne hapla, Bursaphelenchus xylophilus, Heterorhabditis bacteriophora, and Steinernema carpocapsae. H. vermicola had no or weak parasitism on the six assayed nematode species. The phylogenetic trees of ITS region, MAPK gene fragments and their combination revealed that there was genetic variability among species and isolates, but there was no apparent relationship between the genetic variability and their host or geographic origin.