Background: Standard biochemical tests, microscopy, colony characteristics, and mating tests have conventionally been used for the identification of dermatophytes species, but these methods of identification are costly, time-consuming, and require special skills. Objective: Our purpose was to identify a method that enables rapid species identification and strain differentiation of dermatophyte fungi. Methods: We chose 4 restriction enzymes (BsYiI, DdeI, HinfI, and MvaI) that could produce different fragment patterns after enzyme digestion according to species or strain. We performed enzyme digestions after polymerase chain reaction amplification of internal transcribed spacer region and identified different restriction fragment length polymorphisms (RFLP) according to species and strains. Results: All the species included in this study could be easily differentiated using any combination of 2 different restriction enzymes except Trichophyton rubrum and T raubitschekii, which produced identical digestion patterns after all 4 restriction enzyme digestions. In the case of T mentagrophytes, MvaI and DdeI each produced 2 distinct RFLP patterns. Conclusion: This study showed that internal transcribed spacer region analysis using polymerase chain reaction-RFLP through DdeI and MvaI is useful for rapid identification of the majority of dermatophytes species. However, there were 2 different band patterns by DdeI and MvaI restriction enzyme digestion and no correlations between morphologic types and RFLP patterns in T mentagrophytes. (J Am Acad Dermatol 2003;48:857-65.)