Background and purpose: Tuberculosis is among the leading causes of death from infectious diseases in the world. According to World Health Organization (WHO), the estimated rate of TB in Iran was 21 per 100,000 populations in 2015. The present study was designed to evaluate the genetic diversity and transmission dynamics of Mycobacterium tuberculosis in Tehran, Iran. Materials and methods: A total of 80 isolates of Mycobacterium tuberculosis was cultured from TB patients attending a teaching hospital in Tehran, Iran from January 2015 to December 2016. Standard 15-locus Mycobacterial Interspersed Repeat Units/Variable Numbers of Tandem Repeat (MIRU-VNTR) typing was applied to genotype clinical isolates. Results: There were 78 different VNTR profiles comprising 2 clusters and 76 unique patterns. The Hunter – Gaston discriminatory index (HGDI) was 0.990, indicating a high power of discrimination for MIRU-VNTR typing. The QUB26 and MTUB21 loci were designated as highly discriminative. Conclusion: The high genetic diversity among Mycobacterium tuberculosis isolates suggests that transmission may have been caused by different sources.