Stocking wild fish populations with hatchery-bred fish has numerous genetic implications for fish species worldwide. In the present study, 16 microsatellite loci were used to determine the genetic effects of nearly three decades of Murray cod (Maccullochella peelii peelii) stocking in five river catchments in southern Australia. Genetic parameters taken from scale samples collected from 1949 to 1954 before the commencement of stocking were compared with samples collected 16 to 28 years after stocking commenced, and with samples from a local hatchery that supplements these catchments. Given that the five catchments are highly connected and adult Murray cod undertake moderate migrations, we predicted that there would be minimal population structuring of historical samples, whereas contemporary samples may have diverged slightly and lost genetic diversity as a result of stocking. A Bayesian Structure analysis indicated genetic homogeneity among the catchments both pre- and post-stocking, indicating that stocking has not measurably impacted genetic structure, although allele frequencies in one catchment changed slightly over this period. Current genetic diversity was moderately high (H-E = 0.693) and had not changed over the period of stocking. Broodfish had a similar level of genetic diversity to the wild populations, and effective population size had not changed substantially between the two time periods. Our results may bode well for stocking programs of species that are undertaken without knowledge of natural genetic structure, when river connectivity is high, fish are moderately migratory and broodfish are sourced locally.