DNA from 59 Beauveria bassiana isolates and one B. brongniartii isolate were subjected to inter-simple-sequence-repeat-anchored polymerase chain reaction (ISSR-PCR) amplification with three short, 14–18-nucleotide primers that included tandemly repeated sequences. ISSR-PCR was found to be highly reproducible since DNA amplification from several single-spore-isolate subcultures of the B. bassiana F-263 isolate resulted in identical banding patterns. Each primer produced highly polymorphic bands, and high gene diversities for each primer were estimated with a range from 0.24 to 0.28. Consequently, all B. bassiana isolates, including F-263, were characterized by unique banding patterns. A dendrogram created by unweighted pair group method analysis (UPGMA) of ISSR-PCR data showed several small clades composed of isolates that originated from the insect order Coleoptera. They were dispersed throughout the dendrogram and did not comprise a large clade. A similar pattern was found for isolates from Lepidoptera and Hymenoptera.