For the simple and rapid detection/identification of major pathogenic fungal species such as Candida albicans, C. tropicalis, C. parapsilosis, C. glabrata and Aspergillus fumigatus, common primers for these species and specific primers for each species, designed on the basis on the genomic nucleotide sequences of the DNA topoisomerase II genes, were prepared and tested for their specificities in PCR amplifications. Twelve specific primers were pooled and designated PsVI. Genomic DNAs were amplified by the common primer pair, and followed by PCR amplification using PsVI. Using PsVI, six unique DNA fragments, all of which corresponded to a Candida or A. fumigatus species, were specifically and acceptably amplified from each template DNA even in the presence of other DNAs. Similarly, the results of identification of clinical samples based on the PCR amplification coincided with those of conventional identification techniques. The sensitivities of the direct PCR and the nested PCR using PsVI were found to be 1,000 and 50 yeast cells, respectively.