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Tuberculosis (TB) remains a major public health challenge, particularly in sub-Saharan Africa, where the emergence and spread of drug-resistant Mycobacterium tuberculosis (MTB) strains threaten current control efforts. This study aimed to characterize the frequency and distribution of genetic mutations associated with resistance to rifampicin, isoniazid, ethambutol, fluoroquinolones, and second-line injectable agents in MTB isolates from culture-positive pulmonary TB patients in the Republic of Congo. A total of 45 MTB culture-positive samples collected between 2018 and 2019 were analyzed using targeted next-generation sequencing (MinION). Key resistance associated genes, including rpoB, katG, embB, gyrA, and rrs, were examined. Overall, 48% (22/45) of isolates harbored at least one mutation conferring drug resistance. Drug resistance associated mutations were detected in 48% (15/22) of sequences for rifampicin, mainly driven by Ser531Leu, Asp516Val, and His526Tyr. For Ethambutol, the prevalence was 50% (11/22), mainly associated with Met306Val mutation. Isoniazid represented 40.9% (9/22), primarily linked with Ser315Thr mutation. The floroquinolone represented 9% (2/22) mainly driven by Ala90Val and Asp94Gly. Lastly aminoglycosides resistance counted for 4% (1/22) mainly A1401G mutation. The results underscore the critical need to enhance molecular surveillance and strengthen treatment protocols for drug-resistant TB in the Republic of Congo to effectively combat this evolving health crisis.