Technical Abstract: Beauveria bassiana is naturally present in grasshopper populations of the U.S. Northern Plains. It is often rare in a population, but at times can reach a prevalence of 10-15%. One strain, GHA, is registered in the U.S. for use against grasshoppers as well as other insects. We explored the genotypic variation among 60 B. bassiana isolates obtained in 2000 from different grasshopper hosts in 6 separate geographic locations in North Dakota and Montana, North America. All isolations were made from insects alive at time of collection, but subsequently succumbing to infection. Three non-U.S. isolates (ARSEF4197 from Russia, ARSEF357 from Australia and ARSEF1053 from Brazil) were included for comparison; ARSEF 1053 was used as an out-group in some of our analyses. For analysis we employed the ITS1 and ITS2 region nuclear and ribosomal DNA sequences, using ITS1F and ITS4 primers, and AFLP with EcoR1 and Mse1 restriction enzymes and EcoR1-ACC and EcoR1-AGC extensions. ITS sequencing produced five groups, distinguished by the number of changes in the ITS sequence based on mutations at 20 loci. No ITS groups had any correlation with collection site or acridid host species. The largest group, of 41 isolates, included the nonindigenous ARSEF357 and 4197. The other groups had 11, 6 and 2 isolates respectively, while the Brazilian ARSEF1053 occupied its own group. AFLP yielded 207 polymorphic bands. Genotypic distances among every isolate within any particular ITS group ranged between 0.29 and 0.87. Future studies extending our observations to additional isolates from grasshoppers, and from the soil from the collection sites are planned.