Foundational theories in river ecology such as the River Continuum Concept (Vannote et al. 1980) and the Riverine Productivity Model (Thorpe & Delong 1994) emphasise the importance of downstream changes in ecological character driven by physical gradients in slope, energy, substrate, and basal resources. While these concepts are central to ecological research on the main channel, the notion that the ecological character of floodplain wetlands should be controlled by these longitudinal gradients is considered less. Instead, research on floodplain wetlands tends to focus on the fundamental role of hydroperiod, often broadly summarised by assessments of connection frequency. While hydroperiod is clearly important, the fluxes that occur between the main channel and floodplain wetlands highlight the potential for the longitudinal gradients that influence main channel structure and function to also influence floodplain wetlands. This study examines patterns in ecological character in relation to longitudinal position and connection frequency in 41 wetlands located from the middle to lower reaches of the Border Rivers and Condamine-Balonne River systems in the Northern Murray-Darling Basin. Water chemistry, diatoms, macroinvertebrates and fish were sampled in the wetlands. Water samples were also analysed to detect vertebrate-derived environmental DNA. Results showed that reach was a stronger predictor of patterns for all biotic components tested. This finding demonstrates the limitations of connection frequency as a predictor of ecological character, while also highlighting the influence of longitudinal position on the ecological character of floodplain wetlands.