Two pervasive and fundamental impacts of urbanization are the loss and fragmentation of natural habitats. From a genetic perspective, these impacts manifest as reduced genetic diversity and ultimately reduced genetic viability. The growling grass frog (Litoria raniformis) is listed as vulnerable to extinction in Australia, and endangered in the state of Victoria. Remaining populations of this species in and around the city of Melbourne are threatened by habitat loss, degradation and fragmentation due to urban expansion. We used mitochondrial DNA (mtDNA) and microsatellites to study the genetic structure and diversity of L. raniformis across Melbourne's urban fringe, and also screened four nuclear gene regions (POMC, RAG-1, Rhod and CRYBA1). The mtDNA and nuclear DNA sequences revealed low levels of genetic diversity throughout remnant populations of L. raniformis. However, one of the four regions studied, Cardinia, exhibited relatively high genetic diversity and several unique haplotypes, suggesting this region should be recognized as a separate Management Unit. We discuss the implications of these results for the conservation of L. raniformis in urbanizing landscapes, particularly the potential risks and benefits of translocation, which remains a contentious management approach for this species.