Early identification of Candida isolates to the species level is necessary for effective antifungal therapy, and can also facilitate control of hospital infections. Phenotype-based methods for identifying Candida species are often difficult and time-consuming. Molecular biological techniques provide a useful alternative approach. In the present study, the ITS1-5.8S-ITS2 regions of fungal rRNA genes were amplified with universal primers in 20 standard strains. Digestion of the PCR products with one restriction enzyme, MspI, allowed discrimination of medically important Candida species, including C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, and C. guilliermondii. Using this method, we successfully identified 137 clinical isolates of Candida. Among them, C. albicans was identified as the most common species, followed by C. parapsilosis, C. tropicalis, C. glabrata, C. krusei, and C. guilliermondii. This method is a simple, rapid, and cost-effective method for differentiation between species that is applicable in clinical laboratories.