AbstractBACKGROUNDBaculoviruses are ideal biological insecticides, providing long‐lasting pest control and environmental benefits. Alphabaculovirus mabrassicae stains, with their broad host range, have been effective in agricultural pest management. Various A. mabrassicae isolates (MbMNPV‐CHb1/CTa/K1/QD, MyseMNPV‐Hb, HearMNPV and MacoNPV‐B) have been identified in different hosts. Identifying more effective A. mabrassicae strains with detailed genetic information is crucial for commercial use.RESULTSLaboratory bioassays showed that the median lethal concentration (LC50) of MyseMNPV‐Hb against Mythimna separata was significantly lower than those against Helicoverpa armigera and Spodoptera exigua, but higher than the LC50 of MbMNPV‐CHb1, MbMNPV‐QD and HearMNPV against H. armigera or S. exigua. Comparative genomic analysis revealed significant differences in genomic composition and single‐nucleotide polymorphisms between MyseMNPV‐Hb and the other isolates. A piggyBac‐like element, likely to have been from Alcis repandata (Lepidoptera: Geometridae), was identified in the genomes of these isolates. Eight genes in the A. mabrassicae genomes were found to be under positive selection.CONCLUSIONAlphabaculovirus mabrassicae isolates exhibit different infectivity in various pests, indicating the need for selecting appropriate isolates specific target pests. This study elucidates the genetic factors contributing to the differential infectivity of A. mabrassicae isolates and extends knowledge on its population characteristics. © 2025 Society of Chemical Industry.