African animal trypanosomosis is a major obstacle to the development of more efficient and sustainable livestock production systems in West Africa. Riverine tsetse species such as Glossina palpalis gambiensis Vanderplank are their major vectors. A wide variety of control tactics is available to manage these vectors, but their elimination will only be sustainable if control is exercised following area-wide integrated pest management (AW-IPM) principles, i.e. the control effort is targeting an entire tsetse population within a circumscribed area. In the present study, genetic variation at microsatellite DNA loci was used to examine the population structure of G. p. gambiensis inhabiting two adjacent river basins, i.e. the Comoé and the Mouhoun River basins in Burkina Faso. A remote sensing analysis revealed that the woodland savannah habitats between the river basins have remained unchanged during the last two decades. In addition, genetic variation was studied in two populations that were separated by a man-made lake originating from a dam built in 1991 on the Comoé. Low genetic differentiation was observed between the samples from the Mouhoun and the Comoé River basins and no differentiation was found between the samples separated by the dam. The data presented indicate that the overall genetic differentiation of G. p. gambiensis populations inhabiting two adjacent river basins in Burkina Faso is low (F(ST)=0.016). The results of this study suggest that either G. p. gambiensis populations from the Mouhoun are not isolated from those of the Comoé, or that the isolation is too recent to be detected. If elimination of the G. p. gambiensis population from the Mouhoun River basin is the selected control strategy, re-invasion from adjacent river basins may need to be prevented by establishing a buffer zone between the Mouhoun and the other river basin(s).