Background: Detection of dermatophytes by microbiological method is sometimes problematic and some atypical microscopic or macroscopic morphology are non-detectable. Due to morphological similarity and existing intermediate forms and variants, unequivocally separating these dermatophytes is not always straightforward, and sampling appropriate isolates for research is often troublesome. The aim of this study was to compare and evaluate use of sequencing chitin synthase 1 gene (CHS1) with conventional methods for identifi cation of dermatophytes species and we researched the genetic patterns of samples collected for general phylogenetic analysis. Material and Method:In the primary screening of 250 clinical samples by KOH microscopy method, 64 isolates has been detected as dermatophytes. All samples were cultured and amplifi ed by PCR Method and positive PCR samples have been sequenced. Clinical isolates (64/250) were analyzed by using sequencing gene CHS1 and genotyped by program DNAMAN and MEGA. Result:The all data were compared with the international database of national center for biotechnology information website. Based on reference sequences of different genotype strains, it was noted that most strains of Trichophyton mentagrophytes were misidentifi cations of Trichophyton interdigitale. Conclusion:This research demonstrated that nested PCR and sequencing can be considered as standard method for the diagnosis of dermatophytosis. Also research gives a fi rst result on genetic evolution of the Dermatophytes strains distributing in Iran. It may aid in the creation of a national database that will be a valuable support for further studies.