In South Korea, the Eurasian otter (Lutra lutra (Linnaeus, 1758)), a semi-aquatic carnivore, is found mainly in lower order streams that tend to have a low abundance of preferred prey fish species. To investigate the relationship between resource use and availability, we used DNA barcoding to identify otter diet items in 24 otter spraints (faeces) from 16 sites along the Nakdong River basin from 4 to 6 June 2014. At these sites fish availability was assessed using scoop nets and casting nets. Fish formed the bulk of otter diet, which included also frogs, mammals, and reptiles. By DNA barcoding (success rate: 72.38%), we identified 79 prey items from 105 bone remains. The diet comprised mostly fish, but frogs, mammals, and reptiles were also identified. The fish fauna and otter diet composition differed significantly. Across the study sites, members of the Cyprinidae dominated in netted samples, but occurred less frequently in otter diet. Because most Cyprinidae are fast swimmers, otters also fed on benthic fishes and frogs, suggesting limited foraging flexibility in otters and specialization on more slowly moving prey.