The overwintering ecology of fish in the seasonal ice zone (SIZ) has been unexplored mainly due to methodological limitations. Environmental DNA (eDNA) is an emerging tool to reveal the distribution and species diversity of fish in diverse aquatic environments, thus the application of eDNA technique would be a possible solution for the methodological limitation in SIZ. Therefore, we aimed to detect the overwintering fish community in the ice-covered Saroma-ko Lagoon, located on the Okhotsk Sea coast of Hokkaido and its inflow using eDNA metabarcoding.