Dissolved organic matter (DOM) composition in freshwater ecosystems is influenced by interactions between physical, chemical, and biological processes that are controlled, at one level, by watershed landscape, hydrology, and their connections. Against this environmental template, humans may strongly influence DOM composition. Yet, we lack a comprehensive understanding of DOM composition variation across freshwater ecosystems differentially affected by human activity. Using optical properties, we described DOM variation across five ecosystem groups of the Laurentian Great Lakes Region: large lakes, Kawartha Lakes, Experimental Lakes Area, urban stormwater ponds, and rivers (n = 184 sites). We determined how between ecosystem variation in DOM composition related to watershed size, land use and cover, water quality measures (conductivity, dissolved organic carbon (DOC), nutrient concentration, chlorophyll a), and human population density. The five freshwater ecosystem groups had distinctive DOM composition from each other. These significant differences were not explained completely through differences in watershed size nor spatial autocorrelation. Instead, multivariate partial least squares regression showed that DOM composition was related to differences in human impact across freshwater ecosystems. In particular, urban/developed watersheds with higher human population densities had a unique DOM composition with a clear anthropogenic influence that was distinct from DOM composition in natural land cover and/or agricultural watersheds. This nonagricultural, human developed impact on aquatic DOM was most evident through increased levels of a microbial, humic-like parallel factor analysis component (C6). Lotic and lentic ecosystems with low human population densities had DOM compositions more typical of clear water to humic-rich freshwater ecosystems but C6 was only present at trace to background levels. Consequently, humans are strongly altering the quality of DOM in waters nearby or flowing through highly populated areas, which may alter carbon cycles in anthropogenically disturbed ecosystems at broad scales.