Aim Candida spp. infection commonly occur in immunocompromised patients. Biochemical assay for identification of Candida spp. is time-consuming and shows many undetermined results. Specific detection for antibody, antigen and metabolites of Candida spp. had low sensitivity and specificity. In this study, we developed a rapid diagnostic method, Multiplex-PCR, to identify Candida spp. Methods Five Candida spp. isolates were cultured, identified with germ tube and API® 20 C AUX (BioMerieux® SA) kit. Furthermore, DNA was purified by QIAamp DNA mini (Qiagen®) kit for Multiplex-PCR assay. Results DNA detection limit by Multiplex-PCR assays for C. albicans, C. tropicalis, C. parapsilosis, C. krusei and C. glabrata were 4 pg, 0,98 pg, 0,98 pg, 0,5 pg and 16 pg respectively. This assay was also more sensitive than culture in that Multiplex-PCR could detect 2.6-2.9 x 100 CFU/ml, whereas culture 2.6-2.9 x 102 CFU/ml. Conclusion Multiplex-PCR is much more sensitive than culture and thus, can be recommended as a sens tive and specific assay for identification of Candida spp. © 2010, Faculty of Medicine, Universitas Indonesia. All rights reserved.