Degenerated PCR primers were used to amplify chitin synthase genes from genomic DNA of Metarhizium anisopliae var. anisopliae. Through cloning and sequencing of approximately 600-bp fragments amplified by PCR, we found three genes encoding different types of chitin synthases, designated MaCHS1, MaCHS2, and MaCHS3. Southern blot analysis performed on genomic DNA showed that each of the chitin synthases MaCHS1, MaCHS2, and MaCHS3 is encoded by a single copy gene. Alignment of their deduced amino acid sequences with those of other euascomycetes separated the sequences into three distinct classes. MaCHS1 was identified as a gene for class I chitin synthase, MaCHS2 for class II, and MaCHS3 for class III. The UPGMA dendrogram and phylogenetic tree of the deduced amino acid sequences revealed the taxonomic and evolutionary position of Metarhizium anisopliae var. anisopliae.