Riverine food webs are often laterally disconnected (i.e. between watercourses) in regulated floodplain wetlands for prolonged periods. We compared the trophic structure of benthic resources and consumers (crustaceans and fish) of the three watercourses in a regulated floodplain wetland (the Gwydir Wetlands, Australia) that shared the same source water but were laterally disconnected. The crustaceans Cherax destructor (yabby), Macrobrachium australiense (freshwater prawn), the exotic fish Cyprinus carpio (European carp) and Carassius auratus (goldfish) showed significantly different d13C values among the watercourses, suggesting spatial differences in primary carbon sources. Trophic positions were estimated by using d15N values of benthic organic matter as the base of the food web in each watercourse. The estimated trophic positions and gut contents showed differences in trophic positions and feeding behaviours of consumers between watercourses, in particular for Melanotaenia fluviatilis (Murray–Darling rainbowfish) and M. australiense. Our findings suggest that the observed spatial variation in trophic structure appears to be largely related to the spatial differences in the extent and type of riparian vegetation (i.e. allochthonous carbon source) across the floodplain that most likely constituted part of the benthic resources.