Intraspecific variation of 25 Metarhizium anisopliae var. anisopliae isolates from various insect hosts and geographical origins was studied using RFLP analysis of the rDNA gene complex and the mtDNA. Heterologous and homologous mitochondrial and ribosomal probes were used to produce RFLP profiles of the isolates. The rDNA analysis allowed the differentiation of only seven isolates but provided evidence for the possible presence of introns in different sites of the nuclear rRNA-gene-complex. PCR amplification of the ITS1-5.8S-ITS2 rDNA region and the mtDNA regions corresponding to the SrRNA and the LrRNA, followed by restriction analysis of the PCR products underlined the conserved nature of these regions. By contrast, digestion with any one of endonucleases Hae III, Cfo I, Hpa II and subsequent RFLP analysis of the mtDNA, allowed the classification of the 25 isolates in 10 distinct groups. Double digestions using the restriction endonucleases Kpn I and Sac I led to maximum isolate differentiation, producing unique patterns for 16 isolates and subdividing the remainder into four groups of two or three isolates. This indicates the potential of mtDNA RFLP analysis as an excellent tool for fingerprinting M. anisopliae isolates and for studying genetic polymorphism.