Adaptive divergence between marine and freshwater environments is important in generating phyletic diversity within fishes, but the genetic basis of adaptation to freshwater habitats remains poorly understood. Available approaches to detect adaptive loci include genome scans for selection, but these can be difficult to interpret because of incomplete knowledge of the connection between genotype and phenotype. In contrast, genome wide association studies (GWAS) are powerful tools for linking genotype to phenotype, but offer limited insight into the evolutionary forces shaping variation. Here, we combine GWAS and selection scans to identify loci important in the adaptation of complex physiological traits to freshwater environments. We focused on freshwater (FW)-native and brackish water (BW)-native populations of the Atlantic killifish (Fundulus heteroclitus) as well as a population that is a natural admixture of these two populations. We measured phenotypes for multiple physiological traits that differ between populations and that may contribute to adaptation across osmotic niches (salinity tolerance, hypoxia tolerance, metabolic rate, and body shape) and used a reduced representation approach for genome-wide genotyping. Our results show patterns of population divergence in physiological capabilities that are consistent with local adaptation. Selection scans between BWnative and FW-native populations identified genomic regions that presumably affect fitness between BW and FW environments, while GWAS revealed loci that contribute to variation for each physiological trait. There was substantial overlap in the genomic regions putatively under selection and loci associated with the measured physiological traits, suggesting that these phenotypes are important for adaptive divergence between BW and FW environments. Our analysis also implicates candidate genes likely involved in physiological capabilities, some of which validate a priori hypotheses. Together, these data provide insight into the mechanisms that enable diversification of fishes across osmotic boundaries.. CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/254854 doi: bioRxiv preprint first posted online Jan. 27, 2018; 3 Author SummaryIdentifying the genes that underlie adaptation is important for understanding the evolutionary process, but this is technically challenging. We bring multiple lines of evidence to bear for identifying genes that underlie adaptive divergence. Specifically, we integrate genotypephenotype association mapping with genome-wide scans for signatures of natural selection to reveal genes that underlie phenotypic variation and that are adaptive in populations of killifish that are diverging between marine and freshwater environments. Because adaptation is likely manifest in multiple physiological traits, we focus on hypoxia tolerance, salinity tolerance, and metabolic rate; traits that are divergent be...