Malassezia pachydermatis isolates (n=185) from skin sites from dogs (n=30) were characterized genetically and biochemically following in vitro culture. Two regions in the chitin synthase-2 gene (chs-2) and the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA were sequenced, and the phospholipase activity of each isolate was assessed. Three chs-2 (i.e. Ac, Bc and Cc) and eight ITS-1 (i.e. AI1, AI2, AI3, AI4, BI1, CI1, CI2 and CI3) sequence types were defined for all 185 samples. The findings revealed that multiple M. pachydermatis genotypes/subgenotypes could be cultured from healthy dogs or from dogs with single or multiple, generalized skin lesions. Subgenotypes AI1 and BI1 were associated with all skin sites of dogs sampled, whereas subgenotype CI2 was mostly linked to a particular location. Isolates derived from skin lesions showed a significantly higher phospholipase activity compared with those from skin sites with no detectable lesions. Genotype B was mainly cultured from healthy skin; only four isolates (9.3%) had low phospholipase activity, whereas other genotypes/subgenotypes were predominantly associated with skin lesions and had a high phospholipase activity. The results of the present study suggest that the distribution pattern of particular genotypes or subgenotypes of M. pachydermatis on the skin of dogs relates to the affinity of the yeast to the host and to particular skin sites.