Despite decades of research and mitigation efforts, declines in freshwater quality resulting from anthropogenic nutrient input remain a persistent issue worldwide. Canada has the greatest number of freshwater lakes in the world, yet we have a limited understanding of the magnitude and scale at which most lakes have been affected by human activities, namely Land Use/Land Cover (LULC) alterations. In response, the NSERC Canadian Lake Pulse Network has compiled the first nationwide systematic database of Canadian lake quality metrics by surveying 664 lakes across 12 ecozones over three years. To assess the influence of human impact on water quality and its spatial variation across this lake set, we built models quantifying the association between watershed LULC and physiochemical water parameters. We found that agricultural and urban land use explained the greatest proportion of variation in water quality among LULC categories (R2 = 0.20–0.29), although threshold values of water quality metrics (i.e., concentrations of major ions, total phosphorus [TP], total nitrogen [TN], dissolved organic carbon [DOC], and Chlorophyll a [Chl-a]) differed among ecozones. Generally, when the percentage of catchment agricultural land use exceeded 30–35%, we noted significant increases in TP, TN, DOC, and Chl-a concentrations. Similarly, when urban land use surpassed 1–6%, we observed significant increases in ion concentrations and specific conductance. Overall, our study highlights that while drivers of water quality may be similar across regions, management strategies for freshwater ecosystems must consider their geographic context to better predict when and where threshold responses will occur.