In this study, we review the history of proboscideans in the Great Lakes region (Ontario and western New York) in the context of local glacial and vegetational histories. Further, we investigate mammoth (Mammuthus) and mastodon (Mammut) environmental niche partitioning using stable isotope analysis of bone and dentin collagen (δ13Ccol, δ15Ncol) and structural carbonate in tooth enamel bioapatite (δ13Csc, δ18Osc), and demonstrate that stable isotopes can be used to identify non-locals among museum specimens with no contextual records. New radiocarbon dates suggest that Ontario mastodons lived in tundra-like environments as well as their more common spruce forest habitat. Local Ontario/New York mammoths and mastodons consumed 100% C3-plant diets and drank low-18O waters, consistent with colder-than-modern climates and proximity to glacial meltwater sources. Mammoths and mastodons occupied distinct environmental niches, characterized by different oxygen- and nitrogen-isotope compositions and geographical locations. This suggests that direct competition for resources was not a major factor in their local extinction. We suggest that both mammoths and mastodons obtained water from sources formed primarily from precipitation rather than glacial meltwater. We describe how high δ15N values in mammoths could have been caused by a combination of preferences for dry environments, consumption of low-nutrient forage (particularly stems and stalks), coprophagy, geophagy, and dung fertilization. We argue that low δ15N values in mastodons could have been caused by consumption of trees and shrubs (including nitrogen-fixing taxa) and a preference for recently deglaciated landscapes and/or spruce environments. Finally, we raise the possibility that mastodons contributed to the spruce-pine transition by browsing directly on spruce trees.