INTRODUCTION: Mycobacterium tuberculosis superinfection is known to occur in areas with high rates of tuberculosis (TB) and has a significant impact on overall clinical TB management. AIM: We aimed to estimate the superinfection rate in cohorts of drug sensitive and multi-drug resistant tuberculosis (MDR TB) patients from Eastern Europe and the potential role of a second MDR TB strain infecting a patient with active non-MDR TB in treatment outcome. METHODS: The study population included 512 serial M. tuberculosis isolates obtained from 84 MDR- and 136 non-MDR TB patients recruited sequentially at sites in Lithuania, Latvia and Russia in 2011-2013. Strains were genotyped using standardized 24-loci Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) typing. RESULTS: Changes in two or more MIRU-VNTR loci suggesting superinfection were detected in 13 patients (5.9%). We found 4 initially non-MDR TB patients superinfected with an MDR TB strain during treatment and 3 of them had an unsuccessful outcome. CONCLUSIONS: An unsuccessful treatment outcome in patients initially diagnosed with drug sensitive TB might be explained by superinfection with an MDR TB strain. Bacteriological reversion could be indicative of superinfection with another strain. Archiving of all serial isolates and their genotyping in case of culture reversion could support therapeutic strategies in high MDR TB burden settings if resources are available.