Field surveys were undertaken to estimate the spatial and seasonal distribution of the Eurasian otter on a 23 km long section of the Gwangju Stream, a first tributary of the Yeongsan River that flows through Gwangju Metropolitan City, from 2009 to 2011. The distribution of the numbers of spraints and sprainting spots as an index of otter activity were examined from upstream of the southwest valley of Mt Mudeung to downstream of the Yeongsan River confluence. The spraint markings of otters were broadly distributed along the streamline, with higher marking intensity downstream of the Yeongsan River confluence. Otter markings (which totaled 615 spraints and 169 sprainting sites over the course of the study) exhibited seasonal cycles, with a prominent peak in March and November, from the confluence of the Jeungsimsa stream to downstream of the Yeongsan River confluence. These results suggest that Eurasian otters, an endangered species in Korea, are likely to utilize the Gwangju Stream as their foraging habitat, and adapt to an urbanised environment.