Populations of anadromous fish have become landlocked in relatively recent geological history (<14,000 years), but the evolutionary impacts of this altered lifecycle on traits underlying seawater performance have not been established. In order to examine the effects of relaxed selection on seawater traits, anadromous and landlocked Atlantic salmon were reared under identical conditions and examined for differences in seawater performance and its underlying physiological and endocrine control during the time of spring downstream migration. Salinity tolerance, survival and initial growth in seawater were greater in anadromous than in landlocked salmon. Abundance of the seawater isoform of gill Na+/K+-ATPase increased in spring in both strains but was greater in anadromous salmon. Hormones associated with seawater acclimation (adrenocorticotropic hormone, cortisol and growth hormone) increased in spring in both strains but were higher in anadromous salmon, whereas plasma thyroid hormones did not differ. Hypothalamic urotensin I mRNA levels also increased in spring and were higher in the anadromous strain. The results provide evidence that salinity tolerance and associated physiological traits are regulated by seasonal stimulation of the hypothalamic-pituitary-interrenal axis, and that relaxed selection on seawater entry traits has decreased this stimulation in landlocked salmon.