Background and aim: Dermatophytosis (tinea, ringworm) is the infection of skin, hair or nail that is caused by various keratinophilic fungi (dermatophytes). Dermatophytosis is a common infection throughout the world including all parts of Iran. As conventional laboratory procedures for identification of different dermatophytes are slow or lack specificity, more rapid and reliable methods are still required.Materials and methods: Dermatophyte fungi were isolated from patients with dermatophytosis and preliminarily identified by macroscopic and microscopic morphological criteria. Total cellular DNA was extracted from isolates using conical grinder. ITS1-5.8s-ITS2 region of rDNA region was amplified by the universal fungal primers ITS1 and ITS4 and digested with Eco RII enzyme.Results: 650-750 bp band was produced , as expected. Digestion of the PCR products with the restriction enzyme EcoRII produced different electrophoretic pattern and allowed us the identification and differentiation of common pathogenic dermatophytes including Trichophyton rubrum, T. interdigital, T. mentagrophytes, T. tonsurans, T.violaceum, T. schoenleinii, T. verrucosum, M.canis, M.gypseum and Epidermophyton floccosum. Conclusion: It seems that this PCR-restriction enzyme (PCR-RE) profile is a rapid and reliable tool for discrimination of important dermatophytes and can be an applicable method in reference medical mycology laboratories for diagnostic, as well as for large-scale epidemiological purposes.