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Riverine tsetse (Glossina) as Glossina palpalis gambiensis Vanderplank 1949 and Glossina tachinoides Westwood 1850 are the main vectors for African animal trypanosomoses in Burkina Faso. Vector control has been proven efficient in disease containment, but its success is endangered by the reinvasion of tsetse from neighbouring areas. Thus, identifying relic populations can enhance the success rate of vector control efforts. This is currently carried out through microsatellite analysis which is time-consuming and costly. Recently, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry-based analysis has become a routine method in microbial species identification. Owing to the rapidness and cost-effectiveness, this approach has been extended towards species identification of higher organisms such as tsetse. Following the recent experiences in distinguishing two genotypes of Prototheca spp., it is of interest to explore the validity of mass spectrometry for tsetse population differentiation. As a preliminary test, we submitted male and female G. palpalis gambiensis and G. tachinoides from Sideradougou and Folonzo, Burkina Faso (distance 60 km) to matrix-assisted laser desorption/ionisation analysis. The wing samples were utilized for protein extraction and mass spectra in a broad mass to charge ratio (2,000-20,000 kDa) were obtained. Specific peaks appeared to represent species, sex and location. Then, a peak list was extracted, containing the peaks in mass-to-charge ratio by revealing their intensities as well. These lists were used to compute a spectral dendrogram and a principle component analysis which displayed the differences among the samples from the two different regions. The results indicate that this technique can be extended with additional tsetse species, ideally with supporting genomic data, to later assist in designing rational vector control strategies.