Little precise information is available on the systematics, genetics, ecology and epidemiology of yeasts of the genus Malassezia from different animal species. In the present study, one hundred and four isolates of Malassezia (lipid dependent or non-lipid dependent) from dogs were characterized by their chitin synthase 2 gene (CHS2), and the large subunit (LSU) and the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA sequences, and compared genetically with well-defined reference strains of Malassezia pachydermatis and heterologous species, including Malassezia furfur and Candida albicans. For each locus examined, three main sequence types (i.e. A, B and C) represented all of the 104 isolates, which were designated as genotypes A, B and C, respectively. A fourth, minor sequence type was also defined for the ITS-1. The nucleotide differences among genotypes was consistent with the magnitudes of intraspecific variability reported in previous studies. The genetic analysis of the sequence data sets (for individual loci) showed that all Malassezia genotypes clustered (with moderate to strong support) with the reference sequences of M. pachydermatis to the exclusion of the outgroups M. furfur and C. albicans. The present study reveals that multiple genetic variants of M. pachydermatis occur on dogs. The multilocus approach employed herein provides a foundation for future investigations of M. pachydermatis from other animals and humans, and their ecology and epidemiology.