The incidence rates of fungemia caused by Candida guilliermondii have been increasing over the past several years. Although still relatively rare (1.3% of all cases of fungemia in Japan), most cases of C. guilliermondii fungemia occur in patients with cancer or hematological malignancy and their mortality rate is high. As C. guilliermondii tends to be resistant to various antifungal agents, early identification of this pathogen and treatment with an appropriate antifungal agent are required to improve survival rates in these patients. However, it is extremely difficult to differentiate C. guilliermondii (Pichia guilliermondii) from members of the C. famata complex. To date, identification based on DNA sequencing has been the only reliable method for the identification of fungal groups. Here, we used a polymerase chain reaction (PCR)-based method that we developed for the simple and reliable identification of C. guilliermondii (P. guilliermondii). A pair of specific primers was designed corresponding to the 18S rDNA sequence. The PCR system was applied to isolates from fungemia patients. These yeasts could not be identified with CHROMagar Candida, but were successfully identified using this PCR-based system.