Genetic variability of 95 isolates of Beauveria bassiana from different geographical regions and hosts was assessed using RAPDs and ITS-RFLP molecular markers in order to characterize its genetic variability. Clusterings were obtained using UPGMA and Principal Coordinates Analysis. Four groups were identified with RAPDs. A 930 bp fragment was amplified with primers ITS1 and PN16, and polymorphisms were observed with AluI and MspI, generating two main clusters. Results indicate low genetic variability present in the Colombian B. bassiana population, no association to host type or geographic locality, and a population structure that is not clonal. This characterization sets criteria for determination of bio-insecticide potentiality, introduction of foreign strains and improvement of entomopathogens within an IPM program.