Aquatic macrophyte communities were assessed in 53 lakes in Wisconsin, U.S.A. along environmental and land use development gradients to determine effects human land use perturbations have on aquatic macrophytes at the watershed and riparian development scales. Species richness and relative frequency were surveyed in lakes from two ecoregions: the Northern Lakes and Forests Ecoregion and the Southeastern Wisconsin Till Plain Ecoregion. Lakes were selected along a gradient of watershed development ranging from undeveloped (i.e., forested), to agricultural to urban development. Land uses occurring in the watershed and in perimeters of different width (0–100, 0–200, 0–500, and 0–1000 m from shore, in the watershed) were used to assess effects on macrophyte communities. Snorkel and SCUBA were used to survey aquatic macrophyte species in 18 quadrats of 0.25 m² along 14 transects placed perpendicular to shore in each lake. Effects of watershed development (e.g., agriculture and/or urban) were tested at whole-lake (entire littoral zone) and near-shore (within 7 m of shore) scales using canonical correspondence analysis (CCA) and linear regression. Overall, species richness was negatively related to watershed development, while frequencies of individual species and groups differed in level of response to different land use perturbations. Effects of land use in the perimeters on macrophytes, with a few exceptions, did not provide higher correlations compared to land use at the watershed scale. In lakes with higher total watershed development levels, introduced species, particularly Myriophyllumspicatum, increased in abundance and native species, especially potamids, isoetids, and floating-leaved plants, declined in abundance. Correlations within the northern and southeastern ecoregions separately were not significant. Multivariate analyses suggested species composition is driven by environmental responses as well as human development pressures. Both water chemistry and land use variables loaded positively with the first CCA axis indicating that these factors are correlated. Land use pressures in Wisconsin are greater in the southeastern portion of the state where lakes have higher concentrations of water chemistry variables including alkalinity, conductivity, pH, calcium, magnesium, and nitrogen. This creates a complex gradient that influences species composition of macrophyte communities from lake to lake.