Longitudinal patterns in dissolved organic carbon (DOC) loads and chemical quality were identified in the Colorado River from the headwaters in the Rocky Mountains to the United States-Mexico border from 1994 to 2011. Watershed- and reach-scale climate, land use, river discharge and hydrologic modification conditions that contribute to patterns in DOC were also identified. Principal components analysis (PCA) identified site-specific precipitation and reach-scale discharge as being correlated with sites in the upper basin, where there were increases in DOC load from the upstream to downstream direction. In the lower basin, where DOC load decreased from upstream to downstream, sites were correlated with site-specific temperature and reach-scale population, urban land use and hydrologic modification. In the reaches containing Lakes Powell and Mead, the two largest reservoirs in the United States, DOC quantity decreased, terrestrially derived aromatic DOC was degraded and/or autochthonous less aromatic DOC was produced. Taken together, these results suggest that longitudinal patterns in the relatively unregulated upper basin are influenced by watershed inputs of water and DOC, whereas DOC patterns in the lower basin are reflective of a balance between watershed contribution of water and DOC to the river and loss of water and DOC due to hydrologic modification and/or biogeochemical processes. These findings suggest that alteration of constituent fluxes in rivers that are highly regulated may overshadow watershed processes that would control fluxes in comparable unregulated rivers. Further, these results provide a foundation for detailed assessments of factors controlling the transport and chemical quality of DOC in the Colorado River.