A total of 162 strains of Hirsutella thompsonii, isolated from infected mites collected worldwide, were examined for the production of Hirsutellin A (HtA). More than half of the broth filtrates exhibited mortality rates superior to 50% when assayed against Galleria mellonella. The presence of the gene coding for HtA, a previously characterized H. thompsonii protein exotoxin, was determined by PCR amplification using gene-specific primers. Most isolates (100 out of 162) were shown to possess the HtA gene. However, the presence of the gene could not be associated with enhanced insecticidal activity. Both isolate groups (with or without an amplifiable HtA gene) produced filtrates that caused the same average mortality rate (65%) when assayed against G. mellonella. The production and secretion of the HtA toxin were estimated by probing broth filtrates with an anti-HtA monoclonal antibody. Again, the detection of the HtA protein was poorly correlated with subsequent mortality rates induced by the broth filtrates of the various H. thompsonii strains. This study suggests that HtA is requisite for neither survival nor pathogenicity, and that H. thompsonii strains are likely to secrete other toxins that have yet to be characterized. Sequencing of a limited number of HtA genes showed that, when present, the gene is highly conserved, and it displays an interesting intronic polymorphism.