Populations of North American river otters (Lontra canadensis) declined throughout large portions of the continent during the early 1900s due to habitat degradation and unregulated trapping. River otters had been extirpated in North Dakota (ND), but the Red River Valley has since been recolonized, with potential source populations including the neighboring states of Minnesota or South Dakota, or the Canadian province of Manitoba (MB). We genotyped 9 microsatellite loci in 121 samples to determine the source population of river otters in the Red River Valley of ND, as well as to assess population structure and diversity of river otters in central North America. Overall, genetic diversity was high, with an average observed heterozygosity of 0.58. Genetic differentiation was low (F ST < 0.05) between river otters in ND and those of Minnesota, suggesting that eastern ND was recolonized by river otters from Minnesota. River otters from MB were genetically distinct from all other sampled populations. Low genetic differentiation (FST = 0.044) between South Dakota and Louisiana (LA) suggested that reintroductions using LA stock were successful. The genetic distinctiveness of river otters from different geographic regions should be considered when deciding on source populations for future translocations.