The Trichophyton mentagrophytes complex comprises a group of fungi that frequently cause dermatophytosis in both humans and animals. Notably, this complex also includes T. mentagrophytes var. interdigitale and T. mentagrophytes var. indotineae among its members. In this study, the RADseq method was employed to investigate the genetic diversity and population structure of 87 T. mentagrophytes isolates collected from human patients in the Czech Republic. This study is the first known application of the RADseq method to investigate the genetic diversity and population structure of dermatophytic fungi. SNP-based computation of the phylogenetic tree and population genetics analysis results in the determination of putative species boundaries. The structure of population genetics in this complex was presented, providing new insights into the genetic diversity and evolution of T. mentagrophytes complex. One specimen was identified as heterozygotic, the hybrid origin is likely resulting from the sexual recombination of two distinct lineages within the T. mentagrophytes complex. This finding highlights the potential importance of sexual recombination in shaping the population structure and evolution of these fungi. Our study showcases how using the RADseq method can effectively analyze the intricate genetic variations in T. mentagrophytes complex populations. At the same time, we emphasize that RADseq is a cost-effective alternative to whole genome sequencing. By utilizing RADseq, our research not only sheds light on the genetic diversity and evolution of the T. mentagrophytes complex but also lays a cornerstone for future epidemiological and pathogenesis studies pertaining to other fungal species.