The quality and quantity of organic matter (OM) in a river system directly affects ecosystem health; thus, managers benefit from an in-depth understanding of the drivers and sources of OM. In the Snake River, a highly altered river-reservoir system in the semi-arid western United States, OM production and loading are key drivers of reservoir anoxia, which leads to several deleterious processes such as mercury methylation. However, sources and quantities of OM to the Snake River, and the effects of impoundment on OM moving through the river-reservoir system, are not well understood. Particulate organic carbon (POC), dissolved organic carbon (DOC), particulate nitrogen (PN), chlorophyll a (chl-a), and δ¹⁵N–PN and δ¹³C–POC isotopic ratios were measured bi-weekly for over 2 years at four locations through the Snake River Hells Canyon Reservoir Complex to determine spatial and temporal patterns of OM quantities and sources. POC concentrations increased through the riverine zone upstream of the reservoirs, likely due to in situ primary production and/or inputs from tributaries and agricultural drains; then decreased through the most upstream reservoir likely due to particle settling. Isotopic ratios and other OM source indicators (δ¹⁵N–PN, δ¹³C–POC, POC:PN, chl-a:POC) show that the dominant source of particulate OM was phytoplankton with seasonal terrestrial/macrophytic inputs. Results highlight the effects of major tributary and agricultural drain inputs, primary production, and impoundment on OM composition and concentration through a large river-reservoir system and may inform water quality management efforts in this and similar systems.