Major drainage divides (separating the water flow draining to different seas) may significantly affect the dispersal, distribution, and genetic structure of semiaquatic animals. Assessing this effect is important for the proper management of both endangered and invasive riparian species. Here, I determined the fine-scale impact of the drainage divide between the Baltic Sea and the Black Sea drainage basins on the genetic structure and dispersal of the Eurasian otter (Lutra lutra) in the Bieszczady Mountains (Poland and Slovakia). I investigated the genetic structure of the otter population and assessed 3 alternative dispersal models using 6 landscape genetics methods. The analyses were based on 48 individual genotypes obtained from 622 fecal and gland secretion samples collected in 2008–2011. Results indicate that the major drainage divide is not a barrier to gene flow for this population. This was established by analyses of population genetic structure and confirmed by analysis of the spatial distribution of samples originating from closely related individuals. In line with these findings, the best-supported dispersal model assumed that otters migrate through mountain passes, away from streams, thus revealing that they are able to cross a drainage divide. The genetic structure of the population studied exhibits an isolation-by-distance pattern; however, the locations of several repeatedly recorded or closely related individuals revealed the occurrence of long-distance movements. Confirmation of the high mobility of otters and their ability to cross a major drainage divide ridge suggest that landscape obstacles and discontinuity of river network are unlikely to stop otter dispersal, gene flow, and recolonization of new areas.