We investigate seasonal variations in the diet and drinking water of four Great Lakes mastodon (Mammut americanum) specimens using stable isotope analysis of serially sampled inner-enamel bioapatite structural carbonate (δ13Csc, δ18Osc), and previously published bulk analyses. Isotopic analyses and thin section measurements showed that mastodon tooth enamel extension rates (~ 12–4 mm/yr, decreasing toward the cervix) were lower than those of mammoths or modern elephants. Mastodons had distinct and highly regular seasonal variations in δ13Csc and δ18Osc, which we interpret in the context of local glacial history and vegetation changes. Seasonal variations in δ18O were large but variations in δ13C were small, and may have been obscured if coarser sampling methods than our inner-enamel sampling approach were used. Thus, our approach may be particularly useful for understanding relatively small seasonal changes in δ13C within C3 environments. The seasonal patterns, though not entirely conclusive, suggest that the Ontario mastodons did not migrate over very long distances. Rather, the climate and seasonal dietary patterns of mastodons within the region changed over time, from ~ 12,400 to 10,400 14C yr BP (~ 15,000 – 12,000 cal yr BP). Insights gained using these methods can contribute to a better understanding of megafaunal extinctions and Paleoamerican lifeways.