Abstract BACKGROUND The fungal genera Metarhizium contain many important multiple species that are used as biocontrol agents and as model organisms for exploring insect–fungal interactions. Metarhizium spp. exhibit different traits of pathogenicity, suggesting that the pathogenesis can be quite distinctive. However, the underlying differences in their pathogenesis remain poorly understood. RESULTS Pathogenicity analysis showed that Metarhizium anisopliae (strain CQMa421) displayed higher virulence against oriental migratory locusts, Locusta migratoria manilensis (Meyen), than the acridid‐specific specie Metarhizium acridum (strain CQMa102). Relative to M. acridum , M. anisopliae possessed a higher conidial hydrophobicity, increased ability to penetrate the host, accelerated growth under hypoxia and enhanced ability for the utilization of different carbon sources. Different distributions of carbohydrate epitopes at cell wall surface of M. anisopliae might also contribute to successful evasion of host immune defenses. Comparative genomics showed that M. anisopliae has 98 more virulence‐related secreted proteins (133) than M. acridum (35), which can be functionally classified as hydrolases, virulence effectors, cell wall degradation and stress tolerance‐related proteins, and helpful to the cuticle penetration and host internal environment adaption. In addition, differences in genomic clusters specifically related to secondary metabolites, including the clusters of Indole‐NRPS hybrid, T1PKS‐NRPS like hybrid, Betalactone, Fungal‐Ripp and NRPS‐Terpene hybrid, may lead to differences in core virulence‐related secondary metabolite genes in M. acridum (18) and M. anisopliae (36). CONCLUSION The comparative study provided new insights into the different infection strategies between M. anisopliae and M. acridum , and further facilitate the identification of virulence‐related genes for the improvement of mycoinsecticides. © 2023 Society of Chemical Industry.