Multidrug-resistant tuberculosis (MDR-TB) is a serious problem in the former Soviet Union and may appear during TB treatment. We aimed to estimate the prevalence of, timing of and factors associated with MDR-TB diagnosis during TB treatment in Moldova, which was part of the former Soviet Union. We analysed data on 3 754 confirmed non-MDR-TB cases (between January 1, 2007 and December 31, 2010) in the Moldovan TB surveillance database, where patients provided sputum specimens for drug-susceptibility testing, multiple times, during treatment. We estimated the percentage of individuals with confirmed baseline non-MDR-TB that were diagnosed with MDR-TB during treatment, documented the time at which MDR-TB was diagnosed, and used a failure-time model to identify factors associated with MDR-TB diagnosis. Between 7.2% and 9.2% of initially non-MDR-TB cases were diagnosed with MDR-TB during treatment. Half of these MDR-TB diagnoses occurred with 3 months of the initial diagnosis. An increased MDR-TB risk during treatment was associated with baseline resistance to first-line TB drugs (linear increase in risk per additional drug), previous incarceration and HIV co-infection. MDR can appear rapidly during TB treatment. Policy considerations should emphasise management during early treatment by increasing ambulatory TB treatment to prevent nosocomial transmission, and ensuring universal rapid diagnostics access to prevent acquisition and transmission of drug resistance.