Background and Objectives: Genetic diversity of Mycobacterium tuberculosis clinical isolates from tuberculosis patients in the multiethnic province of Alborz, Iran was assessed. Materials and Methods: A total of 17 isolates in the period of 2012-2013 were collected and subjected to a Multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) consisted of 6 variable numbers of tandem repeats (VNTRs) including ETR-A, ETR-B, ETR-C, ElTR-D, ETR-E, ETR-F, 5 Mycobacterial Interspersed Repetitive Units including MIRU10, MIRU16, MIRU26, MIRU39, MIRU40, and 1 Queen University of Belfast locus, QUB11. Results: This classified all isolates into 17 distinct MIRU-VNTR types, a reflection of a highly heterogenic population. Within the 12 used VNTR loci, ten proved highly or moderately discriminant according to the calculated HGDI scores. No cluster of isolates was identified in the study panel, giving a clustering rate of 0%, several events of SVL (N=5) and DVL (N=4) and TVL (N=3) were detected. Conclusion: The greater heterogeneity observed here by MLVA-VNTR analysis is most likely due to limited background data in the study region rather than a genuine more heterogeneous population compared to other provinces of the country.