In this study, we examined the temporal and spatial variability of dissolved organic matter (DOM) abundance and composition in the lower Mississippi and Pearl rivers and effects of human and natural influences. In particular, we looked at bulk C/N ratio, stable isotopes (δ ¹⁵N and δ ¹³C) and ¹³C nuclear magnetic resonance (NMR) spectrometry of high molecular weight (HMW; 0.2 μm to 1 kDa) DOM. Monthly water samples were collected at one station in each river from August 2001 to 2003. Surveys of spatial variability of total dissolved organic carbon (DOC) and nitrogen (DON) were also conducted in June 2003, from 390 km downstream in the Mississippi River and from Jackson to Stennis Space Center in the Pearl River. Higher DOC (336−1170 μM), C/N ratio,% aromaticity, and more depleted δ ¹⁵N (0.76−2.1‰) were observed in the Pearl than in the lower Mississippi River (223−380 μM, 4.7−11.5‰, respectively). DOC, C/N ratio, δ ¹³C, δ ¹⁵N, and % aromaticity of Pearl River HMW DOM were correlated with water discharge, which indicated a coupling between local soil inputs and regional precipitation events. Conversely, seasonal variability in the lower Mississippi River was more controlled by spatial variability of a larger integrative signal from the watershed as well as in situ DOM processing. Spatially, very little change occurred in total DOC in the downstream survey of the lower Mississippi River, compared to a decrease of 24% in the Pearl River. Differences in DOM between these two rivers were reflective of the Mississippi River having more extensive river processing of terrestrial DOM, more phytoplankton inputs, and greater anthropogenic perturbation than the Pearl River.