Tuberculosis (TB) remains a global health challenge, particularly in regions with high human immunodeficiency virus (HIV) prevalence. HIV weakens the immune system, alters the natural history of TB disease, and is an important risk factor for both drug-sensitive and drug-resistant forms of TB. However, understanding of the transmission dynamics of various TB lineages in high HIV settings is limited. Additionally, the influence of HIV on the diagnosis of drug-resistant TB and the mutations that confer resistance to anti-tuberculous drugs remain unclear. Using whole genome sequencing (WGS) data from Mycobacterium tuberculosis complex (Mtbc) isolates collected from a population-based TB study in Botswana, we sought to 1) evaluate the diagnostic accuracy of WGS-based method in detecting resistance to first-line anti-tuberculous drugs in HIV-positive and HIV-negative individuals; 2) determine whether Mtbc variants with high-fitness cost mutations occurred more frequently among individuals with HIV-infection; and 3) use Bayesian phylodynamic analysis to comprehensively delineate the spread of Mtbc lineages underlying the TB endemic in Botswana. Between 2012 and 2016, 4,331 participants with presumed TB in the greater Gaborone and Ghanzi districts were enrolled into the research study, of whom 2,162 participants had bacteriologically-confirmed TB. Mtbc were successfully cultured and sequenced from 1,426 participants. WGS was performed with the Illumina NextSeq 500 platform and data were analyzed with MTBseq pipeline. Overall, 1,350 participants had available WGS and culture-based phenotypic drug susceptibility testing (pDST; reference standard) results. Of those, 702 (52.0%) were HIV-positive participants, of whom 228 (32.5%) had advanced immunosuppression (≤200 CD4+ cells/mm3). Culture-based pDST (reference standard) indicated 104 (7.7%), 72 (5.3%), 58 (4.3%), and 27 (2.0%) Mtbc isolates had resistance to isoniazid, rifampin, ethambutol, and pyrazinamide, respectively; 49 (3.6%) had resistance to at least isoniazid and rifampin (multidrug-resistant TB). While the overall distribution of anti-tuberculous resistance-conferring mutations among isolates appeared to be similar between HIV-positive and HIV-negative participants, WGS-based detection may have lower sensitivity for identifying isoniazid resistance among participants who had ≤200 CD4+ cells/mm3 as compared to participants without HIV (37.5% vs. 71.7%; p-value=0.019). Furthermore, of the Mtbc isolates analyzed, we found the predominant lineage in Botswana was L4 (87.8%), followed by L1 (6.0%), L2 (5.3%), and L3 (0.8%). We identified multiple circulating L4 sublineages, with L4.3.4 being the most prevalent (29.5%). Temporally resolved phylogenies inferred the most recent common ancestor (MRCA) to have emerged around year 1727 (95% highest posterior density [HPD], 1607–1828) and 1900 (95% HPD 1854–1938) for L1 and L2, respectively. Time to MRCA varied among L4 sublineages; ranging from 1695 for L4.1.2 (95% HPD 1546–1817) to 1941 for L4.3.2 (95% HPD 1911–1966). Phylodynamic analysis revealed L4.3.4 expanded steadily from late 1800s to early 2000s. Conversely, L1, L4.4, and L4.3.2 showed population trajectories closely aligned with the HIV epidemic in the region. Meanwhile, L2 saw rapid expansion throughout most of the 20th century but declined sharply in early 1990s. Additionally, pairwise genome comparison of Mtbc highlighted differences in clustering proportions due to recent transmission at the sublineage level. These findings emphasize the diverse transmission dynamics of Mtbc lineages and highlight the potential for phylodynamic analysis of routine sequences to refine our understanding of lineage-specific behaviors, thereby enabling tailored public health interventions for more effective tuberculosis control in high burden settings.