Microsatellite markers were utilized to examine the genetic structure of Murray cod Maccullochella peelii throughout its distribution in the Murray–Darling Basin (MDB) of eastern Australia, and to assess the genetic effects of over three decades of stocking hatchery-reared fingerlings. Bayesian analysis using the programme Structure indicated that the species is largely genetically panmictic throughout much of its extensive range, most probably due to the high level of connectivity between catchments. Three catchments with terminal wetlands (the Lachlan, Macquarie and Gwydir), however, contained genetically distinct populations. No stocking effects were detected in the catchments that were genetically panmictic (either because of low genetic power or lack of effects), but the genetically differentiated Gwydir and Macquarie catchment populations were clearly affected by stocking. Conversely, there was no genetic evidence for survival and reproduction of stocked fish in the Lachlan catchment. Therefore, stocking of M. peelii throughout the MDB has resulted in a range of genetic effects ranging from minimal detectable effect, to substantial change in wild population genetic structure.