Introduction: MIRU-VNTR typing and Spoligotyping are the useful molecular tools for TB epidemiology study. Information regarding genetic diversity and tuberculosis (TB) transmission in Upper Myanmar only is scares. Methodology: We determined the genetic diversity of Mycobacterium tuberculosis (Mtb) and TB transmission from Upper Myanmar TB Reference Laboratory, Mandalay Region, including Mandalay (72), Shan (22), Magway (15), Sagaing (13), Nay Pyi Taw (8), Kachin (7), Chin (2) and Kayah (1). One hundred and forty Mtb isolates were genotyped using 24-locus MIRU-VNTR typing and spoligotyping. Lineage classification and TB transmission analysis were performed. Results: 24-locus MIRU-VNTR typing identified 135 unique profiles and two clusters compared to 35 spoligotyping profiles which contained 12 clusters and 23 unique isolates, Beijing (n=100, 71.4%) was found to be prominent lineage by combine two methods. The expected proportion attributable to recent transmission based on clustering rate was 2.1%. One cluster case was more likely to be in MDR patient. Conclusions: Our findings showed Beijing genotypes were dominant in Upper Myanmar. The usage and analysis of 24-locus MIRU-VNTR typing might prove useful for our broader understanding of TB outbreaks and epidemiology than spoligotyping. The genotypic pattern of this combined method suggests that the lower transmission rate may be due to a higher possibility of reactivation cases in Upper Myanmar.