Genetic variation plays a pivotal role in species viability and the maintenance of population genetic variation is a main focus of conservation biology. Threatened species often show reduced genetic variation compared to non-threatened species, and this is considered indicative of lowered evolutionary potential, compromised reproductive fitness, and elevated extinction risk. The southern purple-spotted gudgeon, Mogurnda adspersa, is a small freshwater fish with poor dispersal potential that was once common throughout the Murray–Darling Basin (MDB) and along the central east coast of Australia. Its numbers and distribution have shrunk dramatically in the MDB due to flow alteration, degradation of habitat, decreasing water quality, and introduction of alien species. We used microsatellite DNA markers to assess population structure and genetic variation at both large (i.e. across basin) and fine (i.e. within river catchments) spatial scales using a substantial sampling effort across the species range (n = 579 individuals; 35 localities). The results consistently indicated very low levels of genetic variation throughout, including along the east coast where the species is relatively common. At the broader scale, three highly differentiated groups of populations were found, concordant with previously reported genealogical distinctiveness. Hence we propose each group as a distinct Evolutionarily Significant Unit. We also inferred a minimum of 12 management units in M. adspersa, with no appreciable gene flow between them. Our study discloses findings relevant for both long- and short-term management, as it informs on the geographic context in which conservation priorities should be defined and specifies biological units for population monitoring and translocations.