Japanese, European, and American eels are migratory, and catadromous migration fish share several common features in their life cycles. Panmixia of American and European eels has been demonstrated by several recent population genetic studies using extensive SNPs (single nucleotide polymorphisms) data. Here, we demonstrate that a Japanese eel group from the Kuma River differs from other panmictic Japanese eels, using a population genetic analysis based on 1000 GB of whole-genome sequence data from 84 eel individuals. Even after removal of the potential adapted/selected SNPs data, we obtained consistent results against panmixia. These results indicated non-panmixia of Japanese eels, unlike in the cases of American and European eels. Given the well-defined spawning area of the Japanese eel, the existence of this different group remains unclear. We propose combined processes, including subdivision of the spawning area, philopatry, and preference/avoidance of habitat, to explain the development of this non-panmictic group in the Japanese eel population. These observations may reflect initial stages of sympatric speciation of these eels.