We investigated in-stream dissolved organic nitrogen (DON) production in multiple streams with differing land-use from Michigan and Wyoming. With native vegetation, we predicted that higher primary production in open-canopy Wyoming streams would result in more autochthonous DON production compared to forested Michigan streams. In agricultural and urban catchments, we predicted that riparian clearing and fertilizer use in Michigan would stimulate in-stream DON production, whereas irrigation-based pasture management in Wyoming would not influence in-stream DON production. Background DON concentrations, which ranged from 64-535 µgN/L (mean=194), were significantly higher in Michigan compared to Wyoming (ANOVA, p<0.0001) and in Michigan, were highest in agricultural streams (p<0.04). Using ¹⁵N-nitrate tracer additions, in-stream DON production was detected in ~50% of the 15 streams. DON production rate (k_DONprod) ranged from 0.02-2.27 h^-1 (mean=0.67) and was highest in a Michigan agricultural and a Wyoming reference stream, yet k_DONprod was not related to primary production. Areal in-stream DON production ranged from 0.05-161 μgN/m²/s (mean=30.4) with highest fluxes from a Michigan agricultural and a Wyoming urban stream.  Although land use did not influence DON production directly, our results show that streams of varying land use have dynamic DON pools and autochthonous DON production may be a significant N source.