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Multidrug-resistant tuberculosis (MDR-TB) has become a major threat to the control of tuberculosis globally. Uganda is among the countries with a relatively high prevalence of tuberculosis despite significant control efforts. In this study, the drug resistance of Mycobacterium tuberculosis to rifampicin (RIF) and isoniazid (INH) was investigated among patients diagnosed with pulmonary tuberculosis in Southwestern Uganda. A total of 283 sputum samples (266 from newly diagnosed and 17 from previously treated patients), collected between May 2018 and April 2019 at four different TB diagnostic centres, were assessed for RIF and INH resistance using high-resolution melt curve analysis. The overall prevalence of monoresistance to INH and RIF was 8.5% and 11% respectively, while the prevalence of MDR-TB was 6.7%. Bivariate analysis showed that patients aged 25 to 44 years were at a higher risk of developing MDR-TB (cOR 0.253). Furthermore, among the newly diagnosed patients, the prevalence of monoresistance to INH, RIF and MDR-TB was 8.6%, 10.2% and 6.4% respectively; while among the previously treated cases, these prevalence rates were 5.9%, 23.5% and 11.8%. These rates are higher than those reported previously indicating a rise in MTB drug resistance and may call for measures used to prevent a further rise in drug resistance. There is also a need to conduct frequent drug resistance surveys, to monitor and curtail the development and spread of drug-resistant TB.