Background: Early diagnosis and treatment of tuberculosis (TB) patients are the most effective ways to protect society from this disease. The objectives of this study were to evaluate the molecular epidemiology of mycobacterial strains by repetitive sequence PCR (rep-PCR) using the DiversiLab System method at Kayseri in Turkey. Methods: A total of fifteen clinically sample isolated Mycobacterial strains were identified as M. tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) by the rep-PCR method (bioMerieux), SD (Standard Diagnostics) rapid tests. Genotyping was performed by the rep-PCR method and clonal affinity was researched. Results: Nine of the strains were identified as MTBC and six strains were identified as NTM by the SD rapid test methods. Five of the strains were identified as NTM by the rep-PCR method as two M. scrofulaceum, M. chelonae, M. fortuitum and M. avium complex (MAC). By genotyping with rep-PCR a total of nine clones were identified, which included two main clones (A and B) and seven other clones (C, D, E, F, G, H, I). Conclusions: It was detected that rep-PCR test is useful method and can be used for the molecular epidemiology and for the identification of mycobacterial strains as they are rapid and practical. However, it is necessary to perform new studies for more mycobacterial strains using the methods described herein.