Background. Trichophyton species are considered the most frequent causative and considerable agents in infection concerns. Objective. The existing training designed to conduct the sequencing analysis of nucleotides of ITS1-5.8S-ITS2 for Trichophyton mentagrophytes. Methods. The isolation and identification of pathogenic fungi, Trichophyton mentagrophytes from clinical specimens was done based on the standard morphological and molecular methods. The genomic DNA of fungal isolates were extracted and purified to amplify with primers of 18S rRNA gene for detection and sequencing the nitrogenous bases to define the genetic variation among clinical isolates of Trichophyton mentagrophytes in compared with strains recorded in NCBI GenBank. Results. Eight isolates of Trichophyton mentagrophytes were isolated and identified from clinical specimen of dermatophytosis. The DNA sequencing analysis showed the presence of some genetic variation in nitrogenous bases between the local isolates and that recorded in locus of world NCBI GenBank. Multiple Alignment sequence using NCBI BLAST revealed the recoding a new mutant isolate among the local isolates based on DNA homology percent. Conclusion. The present results proved the successful the use of molecular methods in diagnosis the fungal isolates of Trichophyton mentagrophytes especially the sequencing analysis to find the genetically bases of these isolates.