When two closely related species migrate to divergent spawning sites, divergent use of spawning habitats can directly reduce heterospecific mating. Furthermore, adaptations to divergent spawning habitats can promote speciation as a by-product of ecological divergence. Here, we investigated habitat isolation and ecological divergence between two anadromous forms of threespine stickleback (Gasterosteus aculeatus), the Japan Sea and Pacific Ocean forms. In several coastal regions of eastern Hokkaido, Japan, these forms migrate to the same watershed to spawn. Our field surveys in a single watershed revealed that segregation of distinct spawning sites between the two forms was maintained within the watershed across multiple years. These spawning sites diverged in salinity and predator composition. Morphological and physiological divergence between the forms also occurs in the direction predicted by ecological differences between the spawning sites. Our data indicate that migration into divergent spawning habitats can be an important mechanism contributing to speciation and phenotypic divergence in anadromous fishes.