In Australia, many species of freshwater fish have rapidly declined following European settlement in the late eighteenth century. The freshwater catfish (Tandanus tandanus) is listed as threatened in Victoria and accordingly, broodstock management and a captive breeding program to facilitate the reintroduction of hatchery bred fish into depleted populations have been suggested. Little work has been conducted on Victorian populations of T. tandanus, despite its threatened status. This study assessed the genetic diversity and genetic structure of T. tandanus in Victoria, using mitochondrial and microsatellite markers. Genetic diversity of T. tandanus in Victoria varied greatly between sites, with the Mallee containing the highest diversity at both markers. Sites where T. tandanus had been introduced in the past typically contained lower measures of genetic diversity, with the exception of the Wimmera site for microsatellite markers. Populations could be assigned to one of four broodfish zones in Victoria, based on levels of genetic differentiation; (1) Mallee, Little Murray, Wimmera, Gum Lagoon and Safe Lagoon; (2) Loddon and Avoca; (3) Turners Lagoon and Phyland Lagoon; and (4) Goulburn. These results will assist managers with implementing broodstock management and a captive breeding program by identifying which sites should be sourced for broodstock and where to reintroduce their progeny, without compromising genetic variation or structure in T. tandanus populations in Victoria.