Background: Tuberculosis (TB) is a preventable and usually curable disease. Yet in 2022, TB was the world’s second leading cause of death from a single infectious agent, after coronavirus disease (COVID-19)1. Aim: By reviving strains isolated at specific years over a 10-year interval and performing next-generation sequencing, we can analyze their strain genotype, epidemiology, drug resistance, and dynamicsTherefore, this study was conducted to examine the historical trends and dynamics of strain genotype, variants, and drug resistance of tuberculosis preserved in the culture bank. Materials and Methods:: Using a retrospective, laboratory-based research approach, 200 strains were randomly selected from over 1,000 diagnostic isolates preserved in the NTRL culture collection from 2010 and 2020. Whole-genome sequencing (WGS) was performed using GridION from Oxford Nanopore Technologies (ONT, Oxford, UK) to analyze these strains. The FastQ file was submitted to the International Mycobacterial Database. Strain genotypes, subtypes, gene mutations of drug resistance, and resistance profiles were identified using TBprofiler, MTBseq, IQ-Tree (version 1.6.12), and EPI2me software. Results: Of the tuberculosis strains selected for the study, 66.5% were from eight out of nine districts of Ulaanbaatar, while 33.5% were sampled from 16 out of 21 provinces. Out of the strains analyzed, 83.9% (95% CI 78.7–89.1) belonged to lineage 2 or the Beijing genotype, while 16.1% (95% CI 10.9–21.3) were lineage-4 or Euro-American genotype. While the proportion of Beijing lineage strains was slightly higher and the Euro-American lineage strains slightly lower in rural populations compared to urban populations, the difference in strain distribution between urban and rural areas was not statistically significant (p=0.485). Among the Beijing lineage strains, only the modern Beijing sublineage (100%) was identified. In contrast, the Euro-American lineage exhibited various sublineages: 4 (0.5%), 4.5 (1%), 4.1.2.1 (Haarlem, 3%), Latin American-Mediterranean (LAM, 7.5%), mainly T (3.5%), and S type (0.5%). Notably, the proportion of Lineage 2 strains increased from 80% in 2010 to 86% in 2020. The overlap of the confidence intervals for 2010 (72.16%–87.84%) and 2020 (79.20%–92.80%) indicates that there has been no significant change in the distribution of Mycobacterium tuberculosis lineages over time. The study revealed that among the selected Mycobacterium tuberculosis strains, resistance rates to first-line anti-tuberculosis drugs were as follows: isoniazid (39%), rifampicin (21%), ethambutol (19%), and streptomycin (34%). Genotypic analysis indicated that the Beijing lineage was predominantly associated with drug-resistant tuberculosis cases, including multidrug-resistant (MDR), poly-drug-resistant, and mono-drug-resistant TB. Notably, the Beijing lineage accounted for 100% of pre-extensively drug-resistant (pre-XDR) TB cases. Within the Haarlem lineage, 33% were MDR-TB. In the Latin American-Mediterranean (LAM) lineage, 13.3% were MDR-TB, 6.6% were poly-drug-resistant, and 13.3% were mono-drug-resistant. Among the mainly T lineage, 42.8% exhibited mono-drug resistance. These findings suggest that the distribution of M. tuberculosis lineages in the Mongolian population has remained relatively stable over time, with no significant temporal changes. Conclusion The distribution of M. tuberculosis genotypes circulating among the population of Mongolia has remained relatively stable over time, with no significant time-dependent changes. Additionally, no mutations associated with resistance to newly introduced anti-TB drugs were detected.