Hortaea werneckii, a black yeast-like hyphomycete that is widely distributed in tropical and subtropical environments, can cause superficial mycotic infection in humans. This fungus was recently isolated from superficial infectious lesions of a guinea pig in Japan. An oligonucleotide primer set specific for Hortaea werneckii was designed on the basis of the internal transcribed spacer regions of the ribosomal DNA (rDNA). Polymerase chain reaction (PCR) with this primer set yielded a 306 bp PCR amplicon from only H. werneckii. This primer set did not amplify DNAs of 42 other related dematiaceous species, including the medically important dematiaceous fungi Cladophialophora carrionii, Fonsecaea pedrosoi, Phialophora verrucosa, and Exophiala dermatitidis, and eight species of medically important yeasts, including Candida (C.) albicans, C. dublinensis, C. glabrata, C. parapsilosis, C. tropicalis, Cryptococcus neoformans var. neoformans, Malassezia furfur, and Trichosporon asahii var. asahii. PCR with this primer set may be a useful technique for rapid identification of H. werneckii.