Water resource development to meet human water needs has altered the natural variability of river flow regimes. The Barwon-Darling River is a dryland river characterized by extreme hydrological variability, which has experienced a change in hydrology and geomorphology since European settlement. We investigated the fish-centered food web structure in six sites in the main river channel to determine if varying levels of hydrological connectivity influenced trophic interactions. Samples were collected in March 2019 during a period of no flows when the river had receded into a series of disconnected waterholes. Analysis was restricted to four species that were caught in high enough abundance at all sites. Stable isotopes (δ13C and δ15N) were used to determine consumer trophic position and community metrics. There were high levels omnivory at all sites and indications of spatial variation with some sites showing more trophic overlap. However, no consistent pattern was found between food web structure and classifications of hydrological character. Due to the extended no-flow period, local habitat characteristics likely had a larger influence on food web structure than antecedent hydrology. Explaining spatial variation among sites likely requires a more thorough assessment of food web structure and site variables than this study allowed.