OBJECTIVES: To estimate the proportion of second-line anti-tuberculosis drug resistance among multidrug-resistant tuberculosis (MDR-TB) patients in Ethiopia. METHODOLOGY: A laboratory-based prospective cross-sectional study was conducted at the National Tuberculosis Reference Laboratory (NTRL), Ethiopia, from February 2022 to July 2024. Phenotypic drug susceptibility testing (pDST) assessed resistance to various second-line anti-tuberculosis drugs. The collected data were entered into Microsoft Excel 2016 and imported into Statistical Package for Social Sciences (SPSS) version 23 for descriptive analysis. RESULT: Of 468 MDR-TB patients, 262 were new, and 206 were previously treated cases. Pre-extensively drug-resistant tuberculosis (pre-XDR-TB) was identified in four (1.52%) new cases and seven (3.40%) previously treated cases. Extensively drug-resistant tuberculosis (XDR-TB) was detected in three (1.15%) new cases and two (0.97%) previously treated cases. Overall, 11 (2.35%) cases were classified as pre-XDR-TB, and five (1.07%) as XDR-TB. Combined resistance to fluoroquinolones (FQs) and bedaquiline were detected in four cases (0.85%), comprising three new cases (1.15%) and one previously treated case (0.49%). Resistance to both FQs and linezolid was detected in a single previously treated case (0.49%) and acquired resistance to second-line drugs was identified in four cases. CONCLUSIONS: Our study showed a prevalence of 2.35% for pre-XDR-TB and 1.07% for XDR-TB among MDR-TB cases, highlighting the importance of continuous surveillance and tailored treatment approaches to control the spread of drug-resistant TB (DR-TB) in Ethiopia. Future studies on MDR-TB surveillance should prioritize the integration of genomic surveillance into routine laboratory-based DR-TB monitoring systems to enhance early detection of resistance patterns, support targeted treatment strategies, and improve overall patient management efforts.