Freshwater tolerance has evolved multiple times among stickleback fishes (Gasterosteidae), such that several closely related species differ in freshwater tolerance. The threespine and fourspine sticklebacks (Gasterosteus aculeatus and Apeltes quadracus) inhabit both freshwater and saltwater, while the blackspotted stickleback (Gasterosteus wheatlandi) and white threespine ecotype (Gasterosteus aculeatus) are largely restricted to saltwater habitats, despite being able to survive acute freshwater exposure as adults. We investigated whether the distribution of blackspotted and white threespine sticklebacks might be limited by a) their capacity to tolerate freshwater during vulnerable early life stages and/ or b) the winter-induced combined stressors of freshwater and cold temperature. The blackspotted stickleback’s fertilization success decreased by 75% in freshwater relative to saltwater, while the threespine and white threespine sticklebacks maintained similar fertilization success in both salinities. Once fertilized, all four species had high survivorship rates (~100%), similar development rates, and comparable metabolic rates (150-250umolO2/um) in freshwater. Overall, our results suggest that reproduction may limit freshwater colonization in this clade, but early life freshwater tolerance does not. In addition, overwintering of adults in freshwater environments may necessitate energetic expenditure that is unsustainable for non-freshwater-colonizing species. Indeed, a cold-induced decrease to their aerobic scope has been shown to occur in the threespine stickleback. To test whether such energetic limitation might restrict non-freshwater-colonizing species’ distributions, we are currently measuring aerobic scopes of threespine and blackspotted sticklebacks who experienced freshwater and saltwater, under winter (4°C) and summer (18°C) temperatures.