Background and Purpose: Dermatophytes as the causative agents of dermatophytosis(ringworm) are widely spread around the world. Accurate identification ofdermatophytes in one area can be particularly important for epidemiological studies.Regarding this, the aim of the present study was to describe the species spectrum ofdermatophytes, isolated from patients in Mashhad city, Iran, using the molecular-basedmethod.Materials and Methods: This study was conducted on 79 dermatophyte isolatesobtained from the human skin, hair, and nail specimens. Species identification wasperformed by the polymerase chain reaction-restriction fragment length polymorphismanalysis of ribosomal DNA internal transcribed spacer regions using MvaI restrictionenzyme.Results: The identified species included Trichophyton mentagrophytes/T. interdigitalespecies complex (n=37, 46.8%), Epidermophyton floccosum (n=12, 15.2%), T. rubrum(n=8, 10.1%), Microsporum canis (n=8, 10.1%), T. violaceum (n=5, 6.3%), T. tonsurans(n=4, 5.1%), Nannizzia gypsea (n=3, 3.8%), T. benhamiae (n=1, 1.3%), and T.verrucosum (n=1, 1.3%). The clinical forms of infection were tinea corporis (n=26,32.8%), tinea cruris (n=22, 27.8%), tinea capitis (n=10, 12.6%), tinea unguium (n=7,9%), tinea manuum (n=6, 8%), tinea pedis (n=5, 6.3%), and tinea faciei (n=3, 3.5%).Conclusion: As the findings indicated, T. mentagrophytes/T. interdigitale speciescomplex had the highest prevalence, and T. benhamiae appeared to be a new emergingagent of dermatophytosis in Mashhad, northeastern Iran.