Malassezia yeasts are associated with several dermatological disorders. The conventional identification of Malassezia species by phenotypic methods is complicated and time-consuming, and the results based on culture methods are difficult to interpret. A comparative molecular approach based on the use of three molecular techniques, namely, amplified fragment length polymorphism (AFLP) analysis, sequencing of the internal transcribed spacer, and sequencing of the D1 and D2 domains of the large-subunit ribosomal DNA region, was applied for the identification of Malassezia species. All species could be correctly identified by means of these methods. The results of AFLP analysis and sequencing were in complete agreement with each other. However, some discrepancies were noted when the molecular methods were compared with the phenotypic method of identification. Specific genotypes were distinguished within a collection of Malassezia furfur isolates from Canadian sources. AFLP analysis revealed significant geographical differences between the North American and European M. furfur strains.The genus Malassezia has received considerable attention in recent years from dermatologists and other clinicians. This group of basidiomycetous yeasts, long known to be the causal agents of pityriasis (tinea) versicolor, is also increasingly being associated with the causation of folliculitis, papillomatosis, and invasive human infections, as well as potential immunogenic triggering of atopic dermatitis, seborrheic dermatitis, and dandruff (6,9,11,14,18,25,26,29). Malassezia species are listed among the new and emerging yeast pathogens (22,24).The genus Malassezia, until recently, was characterized on the basis of rRNA sequences as consisting of seven species, including the lipid-dependent species M. furfur, M. sympodialis, M. globosa, M. obtusa, M. restricta, and M. slooffiae and the lipophilic species M. pachydermatis (12, 13). Recently, two new species have been identified: M. dermatis (39), which was isolated from atopic dermatitis patients, and M. equi (unpublished), a species that was isolated from the skin of horses (32). The latter species has only tentatively been named and still awaits formal description; the present study deals only with the eight described species.Guillot et al. (17) introduced a physiological system based on lipid assimilation and other phenotypic characteristics for identifying the various Malassezia species. This phenotypic system has been used as the conventional method of identification, though in practice, the test results are not always easy to read. Therefore, several research groups have explored the use of molecular techniques, such as pulsed-field gel electrophoresis (3, 4, 36), randomly amplified polymorphic DNA analysis (1, 3), amplified fragment length polymorphism (AFLP) analysis (40), denaturing gradient gel electrophoresis (40), multilocus enzyme electrophoresis (30), sequencing analysis (37), restriction analysis of PCR amplicons of ribosomal sequences (8,15,16,20,28), and chitin synthase ...