Ammonia emissions from oceans are recognized as one of the most significant natural sources of ammonia globally; however, freshwater sources are rarely considered significant. The Great Lakes region, containing the largest network of freshwater lakes in the world, and a significant urbanized population exceeding 20 million, provides a unique opportunity to evaluate the potential for lacustrine surfaces to contribute to regional ammonia levels. In this work, we combine an analysis of 20 years of water quality data from the Great Lakes region and local water measurements near the Greater Toronto Area with the GEM-MACH (Global Environmental Multiscale model – Modelling Air quality and CHemistry) chemical transport model to examine the influence of the Great Lakes on atmospheric ammonia. This analysis demonstrates that while regional ammonia levels are largely controlled by known terrestrial anthropogenic sources, lacustrine emissions increase summertime (July – September) monthly average NH3 levels by 5 – 8% over the largest regional urban centers, with daily increases of up to 10 – 20%, confirming that the Great Lakes represent a regionally significant natural source of ammonia to the atmosphere.