Molecular detection of multidrug-resistant tuberculosis among smear-positive pulmonary tuberculosis patients in Jigjiga town, Ethiopia Mussie Brhane,1 Ameha Kebede,2 Yohannes Petros 2 1Department of Tuberculosis Culture and DST Laboratory, Harar Health Research and Regional Laboratory, Harar, Ethiopia; 2Department of Biology, College of Computational and Natural Sciences, Haramaya University, Haramaya, Ethiopia Background: Molecular methods that target drug resistance mutations are suitable approaches for rapid drug susceptibility testing to detect multidrug-resistant tuberculosis (MDR-TB). The aim of the study was to determine MDR-TB cases and to analyze the frequency of gene mutations associated with rifampicin (RIF) and/or isoniazid (INH) resistance of Mycobacterium tuberculosis among smear-positive pulmonary tuberculosis patients. Methods: Institution-based cross-sectional study design was employed. Sputum specimens were collected, and using a pretested questionnaire, data for associated risk factors for drug resistance were collected from 105 consecutive smear-positive pulmonary tuberculosis patients in Karamara General Hospital. Specimens were transported to Harar Health Research and Regional Laboratory, Harar, where molecular drug susceptibility testing was performed using GenoType® MTBDRplus assay. Results: Of the total 105 sputum specimens, 98 (93.3%) gave interpretable results, in which 67 (68.4%) were new cases and 31 (31.6%) were previously treated cases. Of these, 80 (81.6%) were sensitive to both drugs and 18 (18.4%) were resistant to RIF and/or INH. The prevalences of MDR-TB in total cases, new, and previously treated cases were 10 (10.2%), 3 (4.5%), and 7 (22.6%), respectively. Among the ten total RIF-resistant specimens, eight (80%) had resulted because of absence of rpoB WT8 and presence of MUT3 and in all specimens, the amino acids changed were Ser531Lue. Of the 18 total INH-resistant specimens, 15 (83.3%) had mutations in the katG gene (katG MUT1, Ser315Thr1 ), indicating high-level resistance, while 3 (14.7%) had mutations in the inhA promoter gene (Cys15Thr), indicating low-level resistance. Conclusion: Among the mutations associated with resistance to RIF and INH, the majority were in codon 531 of the rpoB gene and codon 315 of the katG gene. Relatively high prevalence of MDR-TB was observed in the study. Keywords: GenoType®MTBDRplus, isoniazid, multidrug-resistant tuberculosis, rifampicin, smear-positive pulmonary tuberculosis