Over the last few years, new Malassezia species have been found regularly in Japanese subjects. We isolated another new Malassezia species from a Japanese patient with seborrheic dermatitis (SD), and named it M. yamatoensis. In its physiological characteristics and the utilization of Tween by M. yamatoensis is similar to that of M. furfur and M. dermatis. It is distinguished by its growth temperature. To examine the distribution of the microorganism in the skin of patients with SD and atopic dermatitis (AD), and healthy subjects, we applied transparent dressings to the skin, and detected M. yamatoensis DNA using a non-culture-based method that consisted of nested PCR with specific primers. M. yamatoensis DNA was detected from 3 of 31 SD patients (9.7%), 5 of 36 AD patients (13.9%), and 1 of 22 healthy subjects (4.6%). Therefore, M. yamatoensis is a rare member of the cutaneous microflora.