Amplified fragment length polymorphism (AFLP) was used to investigate polymorphism among 43 Hirsutella thompsonii isolates (33 from Thailand) obtained from various mite species. The outgroups were an unidentified Hirsutella isolate along with Hirsutella nodulosa and Hirsutella kirchneri. Phylogenetic analyses of the AFLP data showed significant variation among isolates and the existence of three H. thompsonii clades. We also investigated the isolates using PCR with specific primers for the Hirsutella exotoxin gene Hirsutelin A (HtA) and 18 of these isolates were used for sequencing of the partial beta-tubulin gene. Phylogenetic analyses of beta-tubulin sequences showed two distinct H. thompsonii clades, one of which included AFLP clades I and II. For both markers grouping of the H. thompsonii isolates was not related to either host mite species or geographical origin, although for the HtA gene one clade contained only isolates with no detectable HtA band. These results confirm the high intraspecific polymorphism of H. thompsonii, and maximum likelihood analysis showed no monophyletic group within this species. To refine the taxonomy of this genus other studies should be undertaken using additional molecular markers and several other Hirsutella isolates.