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To elucidate the phyletic relationships among three members of the entomogenous fungal genus, Nomuraea, with an emphasis on N. rileyi.Relationships were evaluated by analysis of the β-tubulin gene and of inter-simple sequence repeats (ISSR). The amplification product of the partial β-tubulin gene was larger for N. atypicola than for N. rileyi, and sequencing of this gene fragment confirmed that N. atypicola possesses approximately 25 more nucleotides than N. rileyi and N. anemonoides. Based on neighbor joining and bootstrap analysis of the partial β-tubulin gene, N. atypicola failed to form a monophyletic grouping with the other two species of Nomuraea. In contrast, the single isolate of N. anemonoides clustered with the N. rileyi isolates, and both taxa grouped with Epichloe typhina (Hypocreales: Clavicipitaceae). Results from this study suggested that N. rileyi and N. anemonoides are closely related to the Clavicipitaceae. In contrast, evidence indicated that N. atypicola is not closely related to this family, and that this taxon is not a Nomuraea. Based on the 83 polymorphic loci of ISSR, it was observed that isolates of N. rileyi from diverse geographical origins were distinctly different from both N. atypicola and N. anemonoides. Considerable heterogeneity was observed in the 18 isolates of N. rileyi tested, and several clusters contained isolates from disparate geographical locations and hosts. However, three isolates from the Philippines (three host species) and three strains isolated from velvetbean caterpillar (Anticarsia gemmatalis) larvae in South America did cluster together. Two other strains from Brazil (isolated from Spodoptera spp.) were distinct from the velvetbean caterpillar isolates from South America.The β-tubulin gene was generally too conserved to resolve intraspecies variability. However, ISSR did identify polymorphisms among the isolates of N. rileyi tested. The results of this study indicate that ISSR may be used as robust molecular markers for studying the population genetics of this entomopathogenic fungus.