Objective To compare the diagnostic performance of the GenoType MRBDRplus assay with the gold standard phenotypic drug susceptibility testing in the detection of drug resistance among culture isolates obtained from patients in Karachi, Pakistan. Design Mycobacterium tuberculosis isolates were obtained from 96 consecutive tuberculosis patients found to have resistance to isoniazid from two health centers in Karachi (January-November 2017). Isolates were tested for drug resistance against rifampin and isoniazid using the MTBDRplus assay. Results were compared with conventional drug-susceptibility testing and the frequency of specific mutations were reported. Results The MTBDRplus assay had a sensitivity for rifampin resistance of 98.8% (95% CI: 93.4–100) and for isoniazid resistance of 90.6% (95% CI: 83.0–95.6). The MTBDRplus assay showed mutations in rpoB in 81 of the 96 (84.4%) isolates. Of the 87 isolates showing resistance to isoniazid via the MTBDRplus assay, 71 (74.0%) isolates had mutations in the katG gene only, 15 (15.6%) isolates had mutations in the inhA promoter region, and 1 (1.0%) showed mutations in both genes. Conclusion The GenoType MTBDRplus assay in Pakistan can identify subgroups at high-risk of having isolates with mutations in the katG and/or inhA genes. Understanding the local burden of these mutations have implications for local diagnostic and treatment guidelines.