In May 2013, we surveyed 25 pools of varying size, habitat type and hydrology along a 450 km section of the Fortescue River of the Pilbara region of northwest Australia. We sought to (i) investigate basin-wide patterns in the distribution of nutrients and stable isotopes, and (ii) to assess if higher trophic levels (fish) reflect pool biogeochemistry. We measured δ2H and δ18O of water, δ13C of dissolved inorganic carbon (δ13C-DIC) and a range of water quality parameters and nutrient concentrations in all pools. Major sources of dissolved organic matter (DOM) into pools were assessed by fluorescence spectrometry. We also measured δ13C and δ15N of primary producers and fish spanning three feeding niches. Preliminary analyses suggest that pools are most similar to each other according to catchment position (Upper versus Lower catchments). We found that more evaporated pool water (more positive δ18O) correlated to higher dissolved organic nitrogen (DON) concentrations (Pearson’s r = 0.72, P < 0.001), and δ13C-DIC (Pearson’s r = 0.70, P < 0.001). However, the δ13C-DIC of surface waters ranged from -5.0 to -12.6 ‰ (mean -10.40 ± 1.56 ‰) indicating that most DIC has been contributed by groundwater. DOM fluorescence components also demonstrate significant variation in sources and concentrations of DOM supporting aquatic food webs. On-going work is assessing how well fish isotopic composition reflects their immediate habitats and whether these measures can be used as proxies for pool connectivity within sub-catchments.