We describe a novel Malassezia species named Malassezia polysorbatinonusus, isolated from a Japanese patient with seborrheic dermatitis. The internal transcribed spacer (ITS) region of the isolate (LSEM 4845T) were only 94.7% identical to those of M. yamatoensis. A phylogenetic analysis of D1/D2 domain of 26S rDNA and β-TUB-encoding gene sequences also revealed that the novel isolate was distinct from the cluster formed by other Malassezia species. Notably, morphological characteristics and molecular analysis indicated that the novel species is most closely related to M. yamatoensis isolated from a Japanese patient with seborrheic dermatitis, although the two species exhibit different physiological characteristics. In contrast to M. yamatoensis, the novel isolate does not metabolize Tweens and can grow at 40 °C. These characteristics have historically distinguished animal Malassezia isolates from those isolated from humans. Therefore, we conclude the novel isolate constitutes a new species, which we designate M. polysorbatinonusus.