The Triatoma brasiliensis genetic structure was analyzed using the Cyt B gene in different geographic locations and ecotopes after a short and long period after insecticide treatment. Four different localities (16-40 km apart) in the state of Paraíba, Brazil, were sampled. Analysis of molecular variance (AMOVA) showed that grouping populations according to the geographic location or ecotope resulted in a higher variance among populations within groups (Phi(SC) ranging from 0.15 to 0.17) than among groups (Phi(CT) ranging from 0.04 to 0.07). The percentage of variation was reduced among populations within groups and increased among groups (Phi(SC) = 0.08, Phi(CT) = 0.16) by grouping 1) the domiciliary populations from each village and 2) all wild populations. These data indicated that T. brasiliensis is genetically structured both ecologically and at a smaller geographic scale for domiciliary populations. Re-infestations after insecticide treatment were composed of distinct populations, pointing to variable population sources for domiciliary infestations.