Malassezia species colonize the skin of normal and various pathological conditions including pityriasis versicolor (PV), seborrhoeic dermatitis (SD) and atopic dermatitis (AD). To elucidate the pathogenic role of Malassezia species in SD, Malassezia microflora of 31 Japanese SD patients was analyzed using a PCR-based, culture-independent method. Nested PCR assay using the primers in the rRNA gene indicated that the major Malassezia species in SD were M. globosa and M. restricta, found in 93 and 74% of the patients, respectively. The detection rate and number of each species varied similarly in SD, PV and healthy subjects (HSs), whereas AD showed higher values. Real-time PCR assay showed that the lesional skin harbored approximately three times the population of genus Malassezia found in nonlesional skin (P<0.05), and that M. restricta is a significantly more common species than M. globosa in SD (P<0.005). Genotypic analysis of the rRNA gene showed that the M. globosa and M. restricta from SD patients fell into specific clusters, and could be distinguished from those collected from HSs, but not from those colleted from AD patients. Our results indicate that certain strains of M. restricta occur in the lesional skin of SD patients.