We have identified ten microsatellite markers in the entomopathogenic fungus Beauveria brongniartii from three genomic libraries enriched for (AAG)n-, (TG)n-, or (TC)n-repeats. The levels of polymorphism of the identified microsatellite loci were assessed in a collection of Beauveria strains originating from different countries, areas, and host insects. Two geographically separated Swiss populations of B. brongniartii originating from European cockchafer (Melolontha melolontha) were also analysed. Microsatellites containing (AAG)n-repeats generally displayed high levels of polymorphism, whereas microsatellites containing either (TG)n- or (TC)n-repeats displayed lower levels of polymorphism. Cluster analysis revealed that strains isolated from M. melolontha larvae, and two strains isolated from Melolontha hippocastani or Amphimallon solstitiale larvae, formed one cluster which was separated from strains isolated from adult M. melolontha and other adult insects. A high degree of biodiversity was detected among B. brongniartii strains of the two separated Swiss populations. Distinct haplotypes were identified in 29 of 35 B. brongniartii strains from population A and in 12 of 28 B. brongniartii strains from population B. The high discrimination power of the identified microsatellites makes them a valuable tool, suited for the characterization and identification of B. brongniartii strains used as biocontrol agents. In addition, they may be applied to monitor biological control strains of B. brongniartii in the field and possibly to investigate their interactions with indigenous B. brongniartii isolates.