SETTING: The true prevalence of multidrug-resistant tuberculosis (MDR-TB) in Ukraine is not known. Available data are a decade old and limited to only one province. OBJECTIVE : To determine the prevalence of MDR-TB among new and previously treated TB cases in Ukraine and explore the risk factors associated with drug resistance. METHODS : A total of 1550 sputum smear-positive pulmonary TB patients were recruited from 40 clusters throughout Ukraine. Sputum specimens were examined using culture, drug susceptibility testing and pncA gene sequencing. RESULT S : The proportion of MDR-TB among new and previously treated TB cases was respectively 24.1% (95%CI 20.7-27.6) and 58.1% (95%CI 52.1-64.1). More than one third (38.0%) of MDR-TB or rifampicin (RMP) resistant cases showed resistance to either a fluoroquinolone (FQ) or a second-line injectable agent or both. Resistance to pyrazinamide and FQs was low in patients with RMP-susceptible TB. Among new TB cases, the odds of MDR-TB were higher among patients who were younger, female and living in south-eastern provinces, as well as among human immunodeficiency virus-positive patients who belonged to a low socioeconomic group. CONCLUS IONS : Our study showed that the burden of MDR-TB in Ukraine was much greater than previously assumed. Urgent actions are needed to prevent further spread of drug-resistant TB in Ukraine. © 2018 The Union.