Abstract: In order to explore the functional genes of Metarhizium anisopliae related to virulence, a T-DNA containing benomyl resistance gene benA3 was transferred into M. anisopliae strain IMI330189 to construct mutant library by using Agrobacterium-mediated transformation method. The toxicity of some mutants to the third instar larvae of Locusta migratoria was tested at a concentration of 10⁸ spores·g^-1 with artificial bait method. The toxicity of mutant 189-1 was slightly shorter with the LT₅₀ 3.5 d compared to the wild strain IMI330189 with LT₅₀ 4.3 d. The mutants 189-4, 189-5, 189-6, 189-10, 189-11, 189-13 and 189-15 almost lost their toxicity to locust, the corrected mortality rates were less than 50%. By constructing mutant library of M. anisopliae strains IMI330189, we obtained some mutants with virulence lack. The study provided a tool to isolate virulent genes and explore the pathogenic mechanism of M. anisopliae.