The isolating nature of freshwater systems may lead to expectations of substantial genetic subdivision among populations of obligate freshwater species. The freshwater fish, Retropinna semoni has a patchy distribution through coastal drainages of Queensland, eastern Australia. We examined the genetic structure and phylogeny of populations of the freshwater fish R. semoni using ten microsatellites loci and mtDNA markers. Ten South east coast Queensland (SEQ) populations and five from Central East Queensland (CEQ) populations were sampled to examine levels of differentiation within and between rivers at near, medium and broad scale. Very high levels of among population structure was observed (Fst = 0.21) and evidence for contemporary migration among rivers was rare and limited to sites within the same river. The present result revealed that Australian smelt populations had a high level of genetic diversity and distinct population structures. We report the existence of three monophyletic matrilineal lineages. High population structure and limited dispersal mean that recolonization of locally extinct populations is only likely to occur from closely situated populations within rivers. Limited potential for recolonization should be considered as an important factor in conservation and management of this species. The fact that the widely spread species show limited dispersal highlights the importance of conservation in both group in Eastern Queensland.