Wetlands are important refugia for many freshwater aquatic biota. As part of the Flinders and Gilbert Agricultural Resource Assessment, hydrological connectivity of a large number of wetlands located in the Flinders floodplain and estuarine environment were investigated and fish assemblages were sampled at selected sites. These two sets of information were combined together to explore key linkages between the timing and duration of wetland connectivity and surveyed fish assemblages. The connectivity assessment was conducted using a floodplain hydrodynamic model (MIKE 21) and fish assemblages were sampled using a backpack electrofisher, combined with gill and seine beach seine nets. Model predicted flood inundation information was combined with land topography data to quantify connectivity. Results show many off-stream wetlands connected with rivers for a short period of time, while others connected only during large floods. Wetlands located in the lower floodplain produced longer duration of connection. Fish data collected here, combined with previous studies show similar trend in terms of number of fish species and total fish catches. It is interesting to note that more fish species were found in wetlands located close to estuarine environments though they had similar connectivity. This is primarily a function of connection between estuarine/marine waters and freshwater river channels. This highlights the importance of maintaining or even enhancing connectivity between floodplain wetlands and river as well as floodplain and estuarine wetlands. This study serves a blue print for future studies examining ecological health and connectivity of floodplain and coastal wetlands in developing Northern Australia.