Accurate and rapid diagnosis of dermatophytosis, a disease whose prevalence has been steadily increased, is important for successful treatment. Current laboratory methods for diagnosing dermatophytosis rely on KOH mount and fungal culture method. However, these methods have low sensitivity and are time-consuming (2~4 weeks to diagnosis). In our previous study, a rapid molecular diagnostic assay (PCR-reverse blot hybridization assay, REBA) was developed to identify the following 6 main species of dermatophytes: Trichophyton rubrum, T. mentagrophytes, T. tonsurans, Microsporum canis, M. gypseum, and Epidermophyton floccosum. However, the REBA required more evaluation to validate its use in clinical examinations. The aim of the present study was to evaluate and validate the ability of the PCR-REBA to successfully identify dermatophytes in clinical isolates from dermatophytosis patients. Both conventional identification methods and the PCR-REBA were used to assess the presence of species of dermatophytes in 148 clinical isolates. The results of the two approaches were compared, and discrepancies between the two approaches were resolved by fungal ITS1 sequence analysis. T. rubrum was the most prevalent dermatophyte identified by conventional identification methods (118/148, 79.7%) and the PCR-REBA (131/148, 88.4%). The overall rate of consistency between conventional identification methods and the PCR-REBA was 79.0% (117/148 samples). Fungal ITS1 sequence analysis showed that PCR-REBA results were correct for 93.5% (29/31) of the discrepant samples. The PCR-REBA is rapid, sensitive, and highly specific compared with conventional identification methods. Thus, the PCR-REBA is a potentially useful tool for identifying dermatophytes in clinical settings.