Tuberculosis (TB) remains a persistent global public health challenge, with the rise of drug-resistant tuberculosis (DR-TB) complicating all the disease control efforts. The World Health Organization (WHO) has advocated for molecular diagnostic techniques, including whole-genome sequencing (WGS), to enhance TB diagnosis and treatment strategies. In this study, we performed WGS analysis on 49 pulmonary tuberculosis isolates from Mexican patients to identify mutations conferring resistance to 11 key antimicrobial agents: four first-line drugs (isoniazid, rifampicin, ethambutol, and pyrazinamide) and 7 second line drugs (fluoroquinolones, ethionamide/prothionamide, amikacin, kanamycin, capreomycin, streptomycin, and bedaquiline). We identified 89 novel variants: 48 in genes previously associated with drug resistance and 41 in genes not previously linked to resistance mechanisms, including potential novel mutations associated with delamanid resistance. Additionally, we detected 31 mutations across three efflux pump superfamilies (ABC, RND, and MFS); all of these variants warrant further investigation regarding their contribution to antibiotic resistance. This analysis represents approximately 10% of Mexico's national variant registry, providing substantial insight into the molecular epidemiology of drug-resistant tuberculosis within the country. The identification of new resistance-associated variants (RAV) from clinical isolates underrepresented in global databases, contributes to develop improved diagnostic tools, optimize treatment regimens, and probably to elucidate antibiotic resistance mechanisms. Specifically, the identification of RAVs for new drugs like bedaquiline, pretomanid, delamanid, and linezolid, which are central to the most recent schemes of treatment (BPaLM, BPaL, BDLLfxC, BLMZ), is key to the improvement of patient outcomes and preventing the emergence of resistance to these critical therapeutic options.