The genetic structure of reservoir populations is a key characteristic in understanding the persistence of infectious diseases in natural systems. In the Highlands of Madagascar, where plague has persisted since 1920, the black rat, Rattus rattus (L., 1758), is the sole species acting as a reservoir of the disease. Ecological surveys have shown a clear correlation between the locations of the plague-persistence area in Madagascar (above 800 m elevation) and the distribution area of one endemic plague vector, the flea Synopsyllus fonquerniei, which is found exclusively on rats living outdoors. This clear habitat segregation has led to the suggestion that R. rattus populations in the central highlands are divided into indoor- and outdoor-dwelling populations. Using eight microsatellite markers, we analysed the genetic structure of R. rattus populations living within a human plague focus in relation to habitat and geographic distance. We found that habitat by itself was not a structuring factor, unlike geographic distance. Nevertheless, the significant genotypic differentiation of R. rattus populations that was found at a fine spatial scale might relate to differences in population dynamics between rats in indoor and outdoor habitats.