AbstractBACKGROUNDBiological control methods involving entomopathogenic fungi like Beauveria bassiana have been shown to be a valuable approach in integrated pest management as an environmentally friendly alternative to control pests and pathogens. Identifying genetic determinants of pathogenicity in B. bassiana is instrumental for enhancing its virulence against insects like the resistant soybean pest Piezodorus guildinii. This study focused on comparative genomics of different B. bassiana strains and gene expression analyses to identify virulence genes in the hypervirulent strain ILB308, especially in response to infection of P. guildinii and growth on hydrocarbon HC15, a known virulence enhancer.RESULTSStrain ILB308 showed the highest number of virulence‐related features, such as candidate virulence proteins, effectors, small secreted proteins and biosynthetic gene clusters. ILB308 also had a high percentage of unique DNA sequences, including six accessory scaffolds. Gene expression analysis at 4 days post inoculation revealed upregulation of known virulence factors, including Tudor domain proteins, LysM motif‐containing proteins, subtilisin‐like proteases and novel genes encoding secreted effectors and heat‐labile enterotoxins. Growth on HC15 led to the upregulation of genes associated with oxidoreductase activity related to cuticular alkane degradation and fermentation metabolism/antioxidant responses in the hemolymph. The low number of known B. bassiana virulence genes points to novel or unknown mechanisms acting on the interaction between P. guildinii and strain ILB308.CONCLUSIONThe presence of accessory genomic regions and unique virulence genes in ILB308 may contribute to its higher virulence. These genes could be considered as potential targets for enhancing fungal virulence through genetic manipulation. © 2025 Society of Chemical Industry.