Stocking of freshwater fish species with hatchery-bred fish is a common response to depleted wild stocks, but may have numerous genetic implications. Murray cod, Maccullochella peelii peelii (Mitchell), have been produced in captivity for wild stocking programs for more than 30 years. The potential genetic impacts of this stocking program on wild populations was investigated by using eight microsatellite markers to determine the parentage of 1380 offspring from 46 separate spawnings collected over three consecutive breeding seasons, and by estimating the effective population size of the broodfish generation through demographic and genetic methods. Results revealed unexpected incidences of polygamous spawnings (both polygyny and polyandry), multiple spawnings by both sexes within a season and repeated matings between pairs of fish across multiple seasons. Furthermore, approximately half of the broodfish failed to spawn at all over the 3-year study period. This likely contributed to the estimated effective population size of around half of the census size, moderate but significant reductions in allelic richness in all three cohorts investigated and a small but significant reduction in heterozygosity in two cohorts. These results allowed us to make recommendations regarding captive husbandry that will maximise genetic diversity of fish intended for stocking.