Rivers play a major role in the transport and processing of dissolved organic matter (DOM). Disturbances that impact DOM dynamics, such as river impoundments and flow regulation, have consequences for biogeochemical cycling and aquatic ecosystems. In this study we examined how river impoundments and hydrologic regulation impact DOM quantity and quality by tracking spatial and seasonal patterns of DOM in a large, regulated river (Klamath River, USA). Dissolved organic carbon (DOC) concentrations decreased downstream and longitudinal patterns in DOC load varied by season. Export of DOM (as DOC) was largely driven by river flow, while DOM composition was strongly influenced by impoundments. Seasonal algal blooms in upstream lentic reaches provided a steady source of algal DOM that was processed in downstream reaches. DOM at upstream sites had an average spectral slope ratio (SR) > 1, indicating algal-derived material, but decreased downstream to an average SR < 1, more indicative of terrestrial-derived material. The increasingly terrestrial nature of DOM exported from reservoirs likely reflects degraded algal material that becomes increasingly more recalcitrant with distance from upstream source and additional processing. As a result, DOM delivered to free-flowing river reaches below impoundments was less variable in composition. Downstream of impoundments, tributary influences resulted in increasing contributions of terrestrial DOM from the surrounding watershed. Removal of the four lower dams on the Klamath River is scheduled to proceed in the next decade. These results suggest that management should consider the role of impoundments on altering DOM dynamics, particularly in the context of dam removal.