Ecosystem processes and biological community structure are expected to change in a relatively predictable manner along fluvial gradients within river basins. Such predictions are heavily based on temperate rivers, and food web variation along fluvial gradients in Mesoamerican rivers has received limited attention. In this study, we analyzed carbon and nitrogen stable isotope ratios of basal carbon sources and dominant consumer species to examine aquatic food web structure along the fluvial gradient of the Monkey River Basin, Belize. Similar to previous studies in other regions, consumer species richness and functional diversity increased along the downstream fluvial gradient, due in part to the addition of estuarine species in lower reaches and increasing diversity of piscivorous species along the gradient. Aquatic food webs in upstream reaches were primarily supported by allochthonous production sources, and in-stream sources increased in importance along the downstream gradient. Our study system traversing the Maya Mountain Marine Area Transect also provided a unique opportunity to test the utility of primary consumer δ15N as an indicator of watershed impacts within a tropical basin with a diverse biota and a different type of agricultural impact than typically studied (i.e. banana plantations vs. tilled row cropping). As expected, primary consumer δ15N at sites draining impacted watersheds was enriched compared to values from forested reference sites. Assessment of primary consumer δ15N may be a feasible option for monitoring watershed impacts on aquatic food webs in service of the ridge-to-reef conservation strategy adopted for this watershed as well as in other tropical river basins. Copyright © 2010 John Wiley & Sons, Ltd.