Murray hardyhead are a threatened native fish species currently limited to four Victorian and four South Australian populations. The reproductive cycle and diet of two isolated populations of Murray hardyhead from Lake Hawthorn and Cardross Basin 1 near Mildura Victoria were examined in this study. Both wetlands are managed for irrigation disposal and are experiencing declining water levels and increasing salinities as a result of improved drainage practices. Analysis of temporal variation in mean gonado-somatic index (GSI) indicated the species has a prolonged spawning season from September to January in both wetlands, with a spawning peak in October/November. Examination of ovaries and mean GSI throughout the breeding season indicated multiple batch-spawning within the breeding season. The diet in both populations consisted mostly of zooplankton with larger fish also feeding on larger prey including dipteran larvae and pupae, ostracods and fish eggs. A greater variety and abundance of food sources was recorded in the gut contents of fish from Cardross Basin 1 than Lake Hawthorn. Seasonal shifts in length frequency distribution and examination of annual increments on otoliths indicate Murray hardyhead appear to be a largely annual species, with the populations examined dominated by 0+ individuals.