Human land use has led to significant changes in the character of dissolved organic matter (DOM) in lotic ecosystems. These changes are expected to have important environmental and ecological consequences. However, high spatiotemporal variability has been reported in previous studies, and the underlying mechanisms remain inadequately understood. This study assessed variation in the properties of stream water DOM within watersheds across a gradient of agricultural land use with grazing pasture lands as the dominant agricultural type in the southeastern United States. We collected water samples under baseflow conditions five times over eight months from a regional group of first- to fourth-order streams. Samples were analyzed for dissolved organic carbon (DOC) concentration, DOM quality based on absorbance and fluorescence properties, as well as DOM biodegradability. We found that air temperature and antecedent hydrological conditions (indicated by antecedent precipitation index and stream water sodium concentrations) positively influenced stream water DOC concentration, DOM fluorescence index, and the proportion of soil-derived, microbial humic fluorescence. This observation suggests that elevated production and release of microbial DOM in soils facilitated by high temperature, in conjunction with strong soil-stream hydrological connectivity, were important drivers for changes in the concentration and composition of stream water DOM. By comparison, watersheds with a high percentage of agricultural land use showed higher DOC concentration, larger proportion of soil-derived, humic-like DOM compounds, and higher DOC biodegradability. These observations reflect preferential mobilization of humic DOM compounds from shallow organic matter-rich soils in agricultural watersheds, likely due to enhanced soil erosion, organic matter oxidation and relatively shallow soil-to-stream flow paths. Display omitted •We assessed DOM in streams across a gradient of agricultural land use.•Temperature and antecedent precipitation increase DOC and soil microbial DOM.•Agricultural land increases DOC, humic DOM and DOM biodegradability.•Land use alters DOM export through erosion, oxidation and shallow flow path.