Otolith microchemistry was used to identify marine- versus freshwater-derived recruitment of three native freshwater fish species that belong to the southern hemisphere Galaxiidae, including two threatened species, in New Zealand’s longest river system, the Waikato River. Water chemistry data for trace elements and 87Sr/86Sr isotope ratios collected from 5 lentic and 10 lotic water bodies throughout the lower river floodplain, representing potential spawning and rearing sites for these fish, were compared to values obtained by laser ablation inductively coupled mass spectrometry (LA-ICPMS) depth-profiling of Young of the Year fish otoliths sampled from 9 lower river catchment sites. Otolith chemical signatures from the larval rearing period indicated that catchment-scale recruitment for two of the species (Galaxias argenteus and G. fasciatus) was driven predominantly by non-diadromous recruitment from riverine lakes. Diadromous recruitment appeared to be more common for G. maculatus, however, non-diadromous specimens were also identified for the first time from a New Zealand river. Reversing outlet flows linked to river stage appeared be crucial for facilitating dispersal of rheotactic non diadromous larvae out of contributing lakes. This study highlights that some waterbodies can supply a disproportionately large number of recruits to maintain and supply fish populations within a wider riverscape, and that lake outflow management is likely to be crucial to sustain this ecologically-important function.