The genus Malassezia was recently revised to include seven species, but the clinical significance of each of these species is not clearly understood. To obtain a better understanding of the contribution of individual Malassezia species to the epidemiology of pityriasis (tinea) versicolor, we used Leeming-Notman medium to culture patient skin specimens showing positive evidence of Malassezia infection in direct microscopy. Isolates were identified on the basis of recently published morphological and physiological tests for distinction of the new species. Identification using recently developed molecular criteria was also carried out for the ambiguous cases. Malassezia species were cultured from 111 cases of pityriasis versicolor in the Canadian province of Ontario. The most frequently isolated species were Malassezia sympodialis, M. globosa and M. furfur which respectively made up 59.4%, 25.2% and 10.8% of the isolated etiological agents. M. globosa was commonly isolated from a small number of pityriasis versicolor specimens obtained from investigators outside Canada. A large number of additional Ontario specimens with positive direct microscopy failed to yield a culture; however, it is suggested that this is consistent with the standard sampling practice of scraping the older portions of pityriasis lesions rather than the margins, where viable fungal cells are most likely to occur.