Aim: To determine the second-line drug susceptibility testing of Mycobacterium tuberculosis isolates from patients in Bayelsa State, Nigeria. Study Design: A cross sectional study was carried out in this research. Place and Duration of Study: Directly Observed Treatment Short-course (DOTS) Centers across Bayelsa State, between March 2020 and November 2021. Methodology: Ethical approval was obtained from Bayelsa State Ministry of Health, Yenagoa, Nigeria. Informations such as age, sex and residential address was obtained with the help of a questionnaire. A total of 100 sputum sample was collected from 100 patients across all the Local Government Areas. Sputum sample decontamination and homogenization was done using the Sodium Hydroxide/N- Acetyl -L- Cysteine Citrate Solution. Sputum samples were cultured on solid Lowenstein Jensen (LJ) Media. All growths on LJ media were confirmed with Ziehl Neelsen staining and Standard Bioline antigen test. Drug susceptibility test was carried out after bacterial DNA was extracted and amplified using Line Probe Assay (MTBDRplus assay ver 2). Results: Out of 100 patients, 15 had confirmed growth of Mycobacterium tuberculosis. All isolate had no form of mutation on gyr gene, meaning 100% of isolates were susceptibility to flouroquinolones. There were also no mutation detected on rrs gene therefore all strains are also susceptible to Kanamycin, Amikasin and capreomycin. Out of the 15 isolates 14 had no mutation on the eis gene while 1% had mutation of WT2 and MUT1. Conclusion: A good percentage of the isolates are susceptible to second line drugs, therefore cases of extensive drug resistance is not common in Bayelsa State, Nigeria.