Humans have altered many floodplain ecosystems around the world by clearing vegetation, building towns and regulating river flows. Studies discerning gene flow and population structure of floodplain-dwelling animals are rare yet are necessary for understanding the effects of human actions on native populations. In south-eastern Australia, the yellow-footed antechinus (Antechinus flavipes) is the only carnivorous marsupial on many lowland floodplains, yet our knowledge of impacts of human activities is limited. The control region of mitochondrial DNA and 11 microsatellite DNA markers were used to explore historic and current gene flow in A. flavipes across and along the Murray River. Simulations were carried out to test different migration models. We found evidence for historic gene flow along and across the river but inferred that small towns and farmland or cleared floodplain sections restricted current gene flow along the river. Populations along the river appear to be isolated, and should be maintained at large enough sizes to avoid genetic problems such as inbreeding depression and loss of evolutionary potential. We also investigated whether 50-year-long maintenance of high water levels for irrigation in summer, at the time of juvenile dispersal, has led to restrictions in gene flow across the river. We found no evidence for restrictions to gene flow across the river and suggest that large floods and dropping tree branches may aid dispersal across the river.