ABSTRACT Bedaquiline is a key drug recommended by the WHO for the treatment of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant (XDR) TB, and its use could potentially shorten treatment duration with improved outcomes. However, resistance against this drug has increased, resulting in serious concerns. Heteroresistance is among the important obstacles to complicate the detection and treatment of MTB drug resistance. We investigated the presence of bedaquiline heteroresistant MTB isolates from Pakistan to highlight their relevance to bedaquiline resistance. Whole genome sequencing of 50 phenotypically bedaquline-resistant and 50 bedaquiline-sensitive isolates revealed 29% bedaquiline heteroresistance in our study. No significant association of patient variables (age, gender, region and history of anti-tuberculosis treatment [ATT]) was found, while drug resistance pattern among MDR + bedaquiline and XDR patterns (OR, 0.53 [0.01–0.26]; P ≤ 0.001 and OR, 0.09 [0.19–0.50]; P = 0.006) were significantly different to bedaquiline heteroresistance. Higher proportion of bedaquiline heteroresistant cases with no history of bedaquiline containing treatment was found. Most bedaquiline heteroresistant strains ( n = 19) were from lineage 3, none of the strain bear mixed lineage, with Rv0678 mutations (95%) being the most prevalent genetic marker. We identified both new mutations (n = 17) and reported mutations (n = 21) that contribute to bedaquiline heteroresistance.The strains with missense variants had the highest percentage of heteroresistance (56%). Bedaquiline heteroresistance is an important indicator of emerging bedaquiline resistance, predominantly observed in previously treated cases without mixed infections, suggesting a higher likelihood of acquired resistance. Our findings accentuate the complexity of bedaquiline heteroresistance and the need for better diagnostic and appropriate therapeutic treatment approaches for drug-resistant TB with bedaquiline-containing regimens. IMPORTANCE This research is decisive as it investigates bedaquiline heteroresistance in Mycobacterium tuberculosis (MTB) isolates from Pakistan, the sixth highest burden country for drug-resistant tuberculosis (DRTB). Bedaquiline is a key drug in the treatment of MDR/XDR-TB, and the emergence of resistance to this drug threatens global efforts to control tuberculosis. Heteroresistance, where drug-susceptible and drug-resistant strains coexist, complicates detection and treatment, potentially leading to treatment failure. By focusing on MTB isolates from Pakistan, this study addresses a critical gap in understanding the prevalence and genetic mechanisms of bedaquiline resistance in a high-burden region. The use of whole genome sequencing (WGS) adds a cutting-edge approach to identifying mutations associated with resistance, offering valuable insights that could inform more effective treatment strategies and public health policies, ultimately contributing to the global fight against drug-resistant TB.