Members of the genus Malassezia are budding yeasts, characterized by a thick cell wall. Recently, these yeasts have received attention as emerging pathogens. They are common commensals on the skin of animals and can become pathogenic under the influence of various predisposing factors. Central to studying their taxonomy, systematics, and ecology and to diagnosis is the accurate identification of species or operational taxonomic units. To overcome the limitations of current phenotypic and biochemical methods of identification, a PCR-coupled SSCP approach, utilizing sequence variation (0.4-33.5%) in short regions (similar to 250-270 bp) of the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA and the chitin synthase-2 gene (chs-2), was assessed for the identification and differentiation of different species/genotypes of Malassezia, characterized previously by DNA sequencing. Genomic DNA samples (n = 30) from Malassezia isolates cultured from canine skin scrapings were assessed by SSCP analysis of the two different genetic loci, and unequivocal delineation between genotypes and species was achieved. This SSCP approach is considered to provide a practical tool for the rapid and reliable genetic characterization of Malassezia genotypes/species from dogs and for investigating their population genetics and ecology. It will also provide a powerful tool for studies of Malassezia isolates from other animal species.