Background: Subtilisin -like proteases are the group of proteases including keratinases found in dermatophytes which de­graded keratin. Determination of the proteases activity of Trichophyton vanbreuseghemii isolates which were obtained from soil and clinical and soil isolates of Microsporum gypseum in Iran and characterization of their genome were aim of present study.Methods: Ezymatic activity was determined by use of chromogenic substrates. The genes, which coded subtilisin-like pro­teases in above-mentioned dermatophytes, was identified and amplified by using specific primers in PCR. Results: The highest yield of enzyme production was observed in only one isolate of T. vanbreuseghemii Ir-84 whereas low enzyme activity was observed in M. gypseum isolates. Homology study of obtained nucleotide as well as amino acid sequences indicated different rates of homology with other subtilisin-like proteases genes in other pathogenic dermato­phytes. Conclusion: Intra-strain differences were observed in production of serine proteinases and molecular characterization of genes encoding such enzymes could be of great interest for studies on pathogenicity and other purposes.