ABSTRACTAlthough the number of multidrug-resistant (MDR) tuberculosis (TB) cases is high overall, a major gap exists in our understanding of the molecular characteristics and transmission dynamics of the MDR Mycobacterium tuberculosis isolates circulating in Bangladesh. The present study aims to characterize the MDR-TB isolates of Bangladesh and to investigate the mode of transmission. A total of 544 MDR-TB isolates were obtained from a nationwide drug-resistant TB surveillance study conducted between October 2011 and March 2017 covering all geographic divisions of Bangladesh. The isolates were characterized using TbD1 deletion analysis, spoligotyping, and mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing. Deletion analysis showed that 440 (80.9%) isolates were the modern type, while the remainder were the ancestral type. The largest circulating lineage was the Beijing type, comprising 208 isolates (38.2%), followed by T, EAI, and LAM with 93 (17.1%), 58 (10.7%), and 52 (9.5%) isolates, respectively. Combined MIRU-VNTR and spoligotyping analysis demonstrated that the majority of the clustered isolates were of the Beijing and T1 lineages. The overall rate of recent transmission was estimated at 33.8%. In conclusion, the MDR M. tuberculosis isolates circulating in Bangladesh are mostly of the modern virulent type. The Beijing and T lineages are the predominant types and most of the transmission of MDR-TB can be attributed to them. The findings also suggest that, along with the remarkable transmission, the emergence of MDR-TB in Bangladesh is largely due to acquired resistance. Rapid and accurate diagnosis and successful treatment will be crucial for controlling MDR-TB in Bangladesh.IMPORTANCE Multidrug-resistant TB is considered to be the major threat to tuberculosis control activities worldwide, including in Bangladesh. Despite the fact that the number of MDR-TB cases is high, a major gap exists in our understanding of the molecular epidemiology of the MDR-TB isolates in Bangladesh. In our study, we characterized and classified the MDR-TB isolates circulating in Bangladesh and investigated their mode of transmission. Our results demonstrated that the MDR M. tuberculosis isolates circulating in Bangladesh are mostly of the modern virulent type. The Beijing and T lineages are the predominant types and are implicated in the majority of MDR-TB transmission. Our findings reveal that, along with the remarkable transmission, the emergence of MDR-TB in Bangladesh is largely due to acquired resistance, which may be due to nonadherence to treatment or inadequate treatment of TB patients. Rapid diagnosis and adherence to an appropriate treatment regimen are therefore crucial to controlling MDR-TB in Bangladesh.