The rise of pre-extensively drug-resistant (pre-XDR) and extensively drug-resistant (XDR) tuberculosis (TB) represents a serious threat to global health. This research investigates resistance patterns of Mycobacterium tuberculosis (MTB) clinical isolates to fluoroquinolones (FQs) and second-line injectable drugs (SLIDs). The samples were collected as part of the Revised National Tuberculosis Control Programme (RNTCP) at Damien Urban TB and Leprosy Center, Nellore, Andhra Pradesh, India. The study further explores genetic mutations linked to drug resistance. A sum of 954 clinical isolates were examined over a year using GenoType MTBDRsl VER 2.0. Among these, 759 isolates were sensitive to both FQs and SLIDs, while 103 exhibited resistance to one or more SLIDs. A total of 86 isolates exhibited resistance to FQs, 11 to SLIDs, and 6 to both. Genetic analysis of the gyrA gene revealed frequent mutations at codons 90, 91, and 94, with the highest occurrence at position 94. Within the 16S rRNA rrs gene, the G1484T mutation was dominant, followed by A1401G. Additionally, alterations in the eis promoter region, especially the C-14T substitution, were observed. Fifteen isolates displayed hetero-resistance, meaning both drug-resistant and drug-susceptible bacterial populations coexisted within the same sample. Furthermore, 31 isolates carried unidentified mutations, emphasizing the genetic complexity of MTB resistance mechanisms. These findings highlight the necessity for continuous surveillance and genetic analysis to better understand and manage drug-resistant TB.