Estuaries are characterized by periodic fluxes in salinity. Many species of North American killifish (Fundulus) are estuarine specialists and harbor euryhaline phenotypes. Three clades within Fundulus independently radiated into freshwater environments and have lost their abilities to tolerate high salinity. We use Fundulus as a comparative model system for studying the physiological and genetic mechanisms that diverge between euryhaline and freshwater species. Four genomes representing the three different clades were selected for genome sequencing to study the physiological and genetic mechanisms that diverge between euryhaline and stenohaline freshwater species.