Candida dubliniensis is a novel Candida spp. that is similar to Candida albicans with respect to several phenotypic characteristics. However, they differ from each other with respect to epidemiology, pathogenesis, and the rapid development of resistance to fluconazole. In the present study, we used a single-enzyme PCR-restriction fragment length polymorphism (RFLP) technique to differentiate C. dubliniensis from C. albicans. The amplified ITS region of C. dubliniensis was digested once using the enzyme B1nI, whereas that of C. albicans remained intact. All standard strains tested were identified successfully by this method. None of 140 clinical isolates identified morphologically as C. albicans were recognized as C. dubliniensis based on their PCR-RFLP pattern. Our PCR-RFLP method easily differentiated C. dubliniensis from C. albicans, and this result was also demonstrated with standard strains.