Adaptation to salinity affects species distributions, promotes speciation, and guides many evolutionary patterns in fishes. To uncover the basis of a complex trait like osmoregulation, genome-level analyses are sensible. We combine population genomic scans with genome expression profiling to discover candidate genes and pathways associated with divergence between osmotic environments. We compared transcriptome sequence divergence between multiple freshwater and saltwater populations of the rainwater killifish, Lucania parva. We also compared sequence divergence between L. parva and its sister species, Lucania goodei, a freshwater specialist. We found highly differentiated single nucleotide polymorphisms (SNPs) between freshwater and saltwater L. parva populations in cell junction and ion transport genes, including V-type H+ ATPase. Between species, we found divergence in reproduction and osmotic stress genes. Genes that were differentially expressed between species during osmotic acclimation included genes involved in ion transport and cell volume regulation. Gene sets that were divergent in coding sequence and divergent in expression did not overlap, although they did converge in function. Like many studies using genomic scans, our approach may miss some loci that contribute to adaptation but have complicated patterns of allelic variation. Our study suggests that gene expression and coding sequence may evolve independently as populations adapt to a complex physiological challenge.