Cisco (Coregonus artedi) are threatened by climate change and lake eutrophication, and their oxythermal habitat can be assessed with TDO3, the water temperature at which dissolved oxygen equals 3 mg·L−1. We assessed the influence of TDO3 on cisco habitat use, genetic diversity, diets, and isotopic niche in 32 lakes ranging from oligotrophic to eutrophic. Results showed that as TDO3 increased, cisco were captured higher in the water column, in a narrower band, with higher minimum temperatures and lower minimum dissolved oxygen. TDO3 was also negatively related to cisco allelic richness and expected heterozygosity, likely driven by summer kill events. Moreover, TDO3 influenced the isotopic niche of cisco, as fish captured deeper were more depleted in δ13C and more enriched in δ15N compared to epilimnetic baselines. Lastly, cisco in high TDO3 lakes consumed more Daphnia, had fewer empty stomachs, and achieved larger body size. Our work identifies specific characteristics of cisco populations that respond to climate change and eutrophication effects and provides a framework for understanding responses of other cold-water species at the global scale.