Throughout the developing world, urban centres with sprawling slum settlements are rapidly expanding and invading previously forested ecosystems. Slum communities are characterized by untended refuse, open sewers and overgrown vegetation, which promote rodent infestation. Norway rats (Rattus norvegicus) are reservoirs for epidemic transmission of many zoonotic pathogens of public health importance. Understanding the population ecology of R. norvegicus is essential to formulate effective rodent control strategies, as this knowledge aids estimation of the temporal stability and spatial connectivity of populations. We screened for genetic variation, characterized the population genetic structure and evaluated the extent and patterns of gene flow in the urban landscape using 17 microsatellite loci in 146 rats from nine sites in the city of Salvador, Brazil. These sites were divided between three neighbourhoods within the city spaced an average of 2.7 km apart. Surprisingly, we detected very little relatedness among animals trapped at the same site and found high levels of genetic diversity, as well as structuring across small geographical distances. Most FST comparisons among sites were statistically significant, including sites <400 m apart. Bayesian analyses grouped the samples in three genetic clusters, each associated with distinct sampling sites from different neighbourhoods or valleys within neighbourhoods. These data indicate the existence of complex genetic structure in R. norvegicus in Salvador, linked to the heterogeneous urban landscape. Future rodent control measures need to take into account the spatial and temporal linkage of rat populations in Salvador, as revealed by genetic data, to develop informed eradication strategies.