We compared lakes and streams in northern Wisconsin to ask how regional patterns of water chemistry differed, and how ecosystem size relates to water chemistry. By applying traditional statistical approaches in novel ways, we identified factors influencing regional chemistry of lakes and streams. Chemistry differed significantly among the two ecosystem types (p = 0.01), and two distinct variable groups were apparent. Almost two thirds of the variation in 16 constituents was associated with concentration gradients related to groundwater inputs (group 1, represented by Ca) and adjacent wetlands (group 2, represented by dissolved organic carbon [DOC]). Largest differences were observed in groundwater-associated variables. Median Ca concentrations in lakes were an order of magnitude lower than in streams (lake = 1.43 mg L−1, stream = 12.45 mg L−1), and indicate that regional lake chemistry is strongly influenced by precipitation or short groundwater flow paths, whereas streams are more affected by groundwater with substantial mineral water contact. In contrast, variables reflecting wetland influence (e.g., DOC) did not differ between streams and lakes. Despite these differences, as lake and stream size increased, their biogeochemical attributes converged, likely reflecting increased diversity of flow paths to larger systems, greater biogeochemical processing, and changes in landscape composition. Our results have identified critical differences (groundwater inputs) and similarities (organic carbon dynamics) between lakes and streams and emphasize the importance of ecosystem size in interpreting these observations at the regional scale. Studies such as this will provide essential pieces of information for regional and global biogeochemical studies of freshwater ecosystems.