Metapopulations occur in fragmented landscapes, and consist of demographically-independent populations connected by dispersal. Nevertheless, anthropogenic habitat fragmentation may be fatal to metapopulations, as it disrupts dispersal and gene flow, and undermines the balance between population extinction and colonization. Understanding the extent to which particular land-use practices disrupt dispersal and gene flow is therefore crucial for conserving metapopulations. We examined the structure and fragmentation of metapopulations of the endangered growling grass frog (Litoria raniformis) in an urbanizing landscape in southern Australia. Population clustering analyses revealed three distinct genetic units, corresponding to the three wetland clusters sampled. Isolation-by-distance was apparent between populations, and genetic distance was significantly correlated with the presence of urban barriers between populations. Our study provides evidence that urbanization fragments metapopulations of L. raniformis. Managers of L. raniformis in urbanizing landscapes should seek to mitigate effects of urbanization on dispersal and gene flow.