Background: Multidrug-resistant tuberculosis (MDR-TB) continues to be a public health problem. Globally in 2019, a total of 465,000 people developed rifampicin-resistant TB (RR-TB), of which 78% had MDR-TB. There is a paucity of evidence on the determinants of MDR-TB in southern Ethiopia. Hence, this study aimed to assess the determinants of MDR-TB in southern Ethiopia. Methods: A hospital-based case-control study was conducted in southern Ethiopia. The cases were all MDR-TB patients attending TB clinics, and controls were all patients who were declared as cured or treatment completed. The cases were selected by consecutive sampling, and a simple random sampling technique was used for controls. Multivariable logistic regression analysis was done to identify determinants of MDR-TB. Adjusted odds ratios (AORs) with 95% confidence intervals (CIs) were computed, and statistical significance was declared at a P-value less than 5%.