The Eurasian otter (Lutra lutra) is widely distributed in Europe and has been down-listed from Vulnerable to Near Threatened on the IUCN Red List in 2004 following successful conservation interventions. River otter recoveries were driven by active and passive protection of wetlands and a decrease in environmental pollutants. In Hungary, although otter populations have been stable in recent decades, there is little information available regarding genetic diversity, population genetic or phylogeographic structure, and potential barriers affecting gene flow. This study, therefore, aimed to determine genetic diversity and structure across the range of this species in Hungary. To achieve this, we analyzed 255 tissue samples collected since 2002, mainly from road-kill incidents, in various regions of Hungary. We found a relatively high level of genetic diversity in Hungarian otter populations (expected heterozygosity: 0.69–0.74; mean number of alleles per locus: 6.8–7.7). At a regional level, we identified 2 distinct genetic clusters corresponding to 2 river basins (Danube and Tisza). We also identified isolation by distance and patterns of genetic differentiation that appear to reflect population divergence of otters that are spatially restricted to one of these 2 river basins. Our results show the strong influence of river networks on population structure and genetic divergence in otters and provide a framework for the development of conservation management plans for otters in Hungary.