Estuaries are characterized by periodic fluxes in salinity. Some 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. We examined 16 estuarine and freshwater species with representation from each of three clades. Fish from all species were acclimated to either brackish or fresh water then exposed to an acute brackish water challenge. Transcriptome data from gill epithelium tissue were collected. To enable multi-species comparisons, reference transcriptome assemblies were generated de novo for each species then used to analyze transcriptional responses to salinity change by clade and physiology.