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The diverse Potamogeton genus includes over 80 species of aquatic macrophytes that occur across a broad geographic range and have variable response to environmental conditions. This study evaluated how environmental and spatial variables structure assemblage composition and species richness of Potamogeton spp. in the US states of Minnesota and Wisconsin. Variation partitioning analysis was used to study the relative contribution of local, climate and spatial variables in explaining assemblage composition and species richness. Models were also developed for sixteen Potamogeton spp. using partial linear regression. Assemblage composition and total species richness were better explained by the pure effects of spatial and local variables as compared to the pure effects of climate variables. However, geographical structuring of variables suggested that species followed a latitudinal gradient that was strongly related to eutrophication and partially related to climate. Models for individual species were similar although some were disproportionately described by specific categories of explanatory variables. For example, invasive Potamogeton crispus was more tolerant of eutrophication than most species and was also described by a strong spatial grouping of lakes near a large urban area. These results suggest that the distribution of Potamogeton spp. is limited by species tolerances to lake variation in local and climate characteristics across spatial gradients, whereas specific species may be more limited by dispersal barriers between lakes with suitable habitat. This analysis is the first regional evaluation of factors related to the distribution of this ecologically important genus and the importance of landscape-level approaches to ecological conservation is emphasized.