Nosocomial transmission of multidrug-resistant tuberculosis (MDR-TB) was ascertained by 24-locus mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and spoligotyping at four hospitals in the Republic of Moldova, a high MDR-TB burden country. Overall, 5.1% of patients with pan-susceptible TB at baseline were identified with MDR-TB during in-patient treatment. In 75% of cases, the MDR-TB strain was genetically distinct from the non-MDR-TB strain at baseline, suggesting a high rate of nosocomial transmission of MDR-TB. The highest proportion (40.3%) of follow-up MDR-TB isolates was associated with the M. tuberculosis URAL 163-15 strain.