Multi-locus denaturing gradient gel electrophoresis (DGGE) analysis was developed to investigate the genotypes of Beauveria bassiana sensu lato. Sensitive tests indicated all isolates with one or more nucleotide differences at EF-1 and Bloc could be distinguished by DGGE except for one pair of strains that differed at four nucleotide positions. Ten, twelve and five genotypes were identified at the EF-1, Bloc and ITS locus, respectively, among seventeen isolates, which together differentiated 13 genotypes. These results demonstrated that multi-locus DGGE is a potentially useful molecular marker for genotyping, identifying and tracking the fates of experimentally released strains of B. bassiana sensu lato. Moreover, by multi-locus DGGE for scanning B. bassiana sensu lato isolates with different multilocus sequences, genetic diversity of B. bassiana sensu lato was effectively investigated with substantially reduced time and cost in subsequent DNA sequencing.