Analysis of otolith strontium isotope ratios 87Sr/86Sr is an increasingly utilised approach for studying fish migration within freshwater and across salinity gradients. A premise of this approach is that freshwater 87Sr/86Sr ratios are temporally stable within a location, allowing variation in otolith 87Sr/86Sr to be interpreted as movement by fish. We analysed surface and groundwater from the Daly River catchment in the wet-dry tropics of Northern Australia over two years. Analyses of otolith 87Sr/86Sr ratios were also conducted for freshwater Sooty grunter (Hephaestus fuliginosus) and the putatively diadromous Ord River mullet (Liza ordensis). Spatial variation in freshwater 87Sr/86Sr ratios was high (range: 0.71612-0.78059) and there was strong seasonality in water 87Sr/86Sr ratios, with highest values in the wet season. Temporal variation in water 87Sr/86Sr ratios is attributed to seasonal patterns in surface run-off from headwaters of ancient geologic origin versus input from groundwater aquifers interacting with younger geological formations. Temporal variation in water 87Sr/86Sr ratios precluded robust inference on movement within freshwater for both species, although movement across salinity gradients by Ord River mullet was clearly identified. We conclude that temporally and spatially replicated water Sr data should be a general requisite for studies that use analyses of otolith Sr (87Sr/86Sr, Sr/Ca, Sr/Ba) to make inferences about fish movement and migration.