Although opportunistic infection including fungal infection is often associated with living donor liver transplantation followed by immunosuppressive therapy, antifungal agents are empirically given to most patients without a definitive diagnosis of fungal infection. Indeed, there is no diagnostic test available, that shows both high sensitivity and specificity for fungal infection. In this study, we developed a polymerase chain reaction(PCR)-based system for rapid diagnosis of fungal infection. This system consisted of two PCR steps: the first PCR for most species of fungi and 4 kinds of nested PCR for Aspergillus or Penicillium (ASP/PEN), Candida glabrata (C. glab), other Candida species including Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida guiliermondii (CAN), and a broad spectrum of fungi (Broad). The newly developed PCR-based system was applied to 28 recipients of with living donor liver transplantation to determine its clinical usefulness in early and differential diagnosis of fungal infection. A total of 514 blood samples from 28 patients ware analyzed. Nested PCR assays were positive in 118 samples from 19 patients: 4 patients (30 samples) with ASP/PEN, 5 patients (29 samples) with C. glab, 12 patients (61 samples) with CAN. All of these samples were positive with nested PCR for Broad as well. Even in samples that were negative on blood culture or ELISA for Aspergillus antigen, nested PCR assays were able to amplify the fungal DNA. Furthermore, all samples positive for Aspergillus antigen tested positive on nested PCR. In conclusion, the PCR-based system we developed was thought to be useful for early diagnosis of fungal infection.