The global tuberculosis (TB) epidemic is becoming progressively more complex due to the increasing prevalence of multidrug-resistant TB (MDR-TB), particularly with resistance to fluoroquinolones (FQs). This study focuses on identifying genetic mutations in the gyrA and gyrB genes of Mycobacterium tuberculosis that drive FQ resistance. Sputum samples from suspected pulmonary TB patients were analyzed using PCR and sequencing to detect mutations within the quinolone resistance-determining regions (QRDR). The analysis revealed that mutations in gyrA, especially S95T, are prevalent and play a key role in FQ resistance. Additionally, less frequent mutations in gyrB, such as E501D and A533P, were also detected. These findings shed light on the molecular mechanisms contributing to FQ resistance in MDR-TB strains and underscore the need for enhanced diagnostic methods to identify resistance patterns more accurately. The insights gained from this research offer a foundation for improving TB treatment approaches and addressing the growing challenge of drug-resistant TB worldwide.