Degenerate PCR primers were used to amplify a 600-bp conserved gene region for chitin synthases from genomic DNA of Sporothrix schenckii, a dimorphic fungal pathogen of humans and animals. Three chitin synthase gene homologs were amplified as shown by DNA sequence analysis and by Southern blotting experiments. Based on differences among the predicted amino acid sequences of these homologs, each was placed within one of three different chitin synthase classes. Phylogenies constructed with the sequences and the PAUP 3.1.1 program showed that S. schenckii consistently clustered most closely with Neurospora crassa in each of the three chitin synthase classes. These findings are significant because the phylogenies support by a new method the grouping of the imperfect fungus S. schenckii with the Pyrenomycetes of the Ascomycota.