Multidrug-resistant tuberculosis (MDR-TB) poses a severe global public health threat, further compounded by the restricted effectiveness of current therapies, protracted treatment schedules, and the risk of serious adverse effects. Prior to the implementation of the current novel treatment regimens globally, the global cure rate for MDR-TB was relatively low. In our study in China, we observed that treatment failure patients, continuously infected with the primary strain, tended to develop phenotypic or genotypic acquired drug resistance when subjected to ineffective treatment. Through conducting whole-genome sequencing on serial strains from treatment failure patients, we discovered that WGS is capable of detecting low-frequency resistance mutations and heterogeneous resistance with remarkable sensitivity. The recent development of bedaquiline and delamanid has revolutionized the treatment landscape for MDR-TB. However, while bedaquiline (BDQ) has enhanced treatment outcomes for MDR-TB patients, its widespread use has given rise to BDQ resistance. As acquired resistance to BDQ continues to increase, offering critical insights for managing MDR-TB. The application of whole-genome sequencing holds great promise for deepening our understanding of drug resistance mechanisms in Mycobacterium tuberculosis. Following the introduction of the new WHO-recommended all-oral short-course regimens, ongoing surveillance of emerging new drug-resistant strains is imperative. Continued innovation, global collaboration, and improved diagnostics are vital for the development of practical, accessible, and affordable MDR-TB treatments.