Background and Objectives: Monitoring of contagious diseases is important to advance our knowledge of their epidemi- ology and to enable more impressive investigation and prevention efforts. This study aimed to examine antifungal drug sus- ceptibility and molecular analysis of clinical isolates of Trichophyton rubrum and Trichophyton mentagrophytes in humans and cattle. Materials and Methods: A total of 400 patients and 500 cattle were evaluated in this study. Dermatophytosis was confirmed in cases by direct microscopy and culture methods. Antifungal drug susceptibility profiles, MIC , and MIC of isolates were 50 90 determined using the broth microdilution method. Multiplex-PCR, RAPD PCR, and sequencing methods were used for the genetic analysis of virulence genes and the ITS1 and ITS2 regions, respectively. Results: A total of 175 patients and 120 cattle were diagnosed with dermatophytosis. Dermatophytes showed a remarkable rate (30%) of terbinafine resistance. T. mentagrophytes showed lower susceptibility than T. rubrum (MIC=16 μg/mL). Strains harboring Mep1, Mep2, and Mep4 genes had the highest frequency among all genotypes. A RAPD-PCR dendrogram divided T. mentagrophytes and T. rubrum strains into three and six groups, respectively. Conclusion: A notable rate of resistance to terbinafine in isolated dermatophytes was reported in this study. Examination of RAPD-PCR results showed that T. rubrum strains had higher genetic diversity than T. mentagrophytes. Genetic monitoring of dermatophytes must be considered an important factor in providing fungal infection prevention and treatment approaches.