Organic matter in soils and sediments derives from a mixture of biological origins, often making it difficult to determine inputs from individual sources. Complicating the determination of source inputs to soil and sedimentary organic matter (OM) is the fact that physical and microbial processes have likely modified the initial composition of these sources. This study focused on identifying the composition of watershed-derived OM to better understand inputs to inland waters and improve our ability to resolve between terrigenous and aquatic sources in downstream systems, such as estuaries and coasts. We surveyed OM sources from the Yuba River watershed in northern California to identify specific biomarkers that represent aquatic and terrigenous OM sources. Multiple classes of organic proxies—including sterols, fatty acids (FA), lignin phenols and stable carbon and nitrogen isotope values (δ13C, δ15N)—were measured in soils, vegetation, charcoal, and freshwater plankton to characterize representative source endmembers. Sterols—including 27-nor-24-cholesta-5,22-dien-3β-ol, cholesta-5,22-dien-3β-ol, 24-methylcholesta-5,22-dien-3β-ol and cholesta-5-en-3β-ol, and positive δ15N values—were associated with aquatic OM (plankton, suspended particulate OM), whereas lignin phenols, long chain FA, and diacids characterized terrigenous sources (soils, charcoal, vegetation). Trends in organic carbon and biomarker signatures in soil samples showed a response to environmental disturbance (i.e., mining, agriculture) through an inverse relationship between OM content and land use. Results from this study demonstrate the utility of multi-biomarker studies for distinguishing between OM from different sources and land uses, offering new insights for biogeochemical studies in aquatic systems.