In West Africa, tsetse species of the subgenus Nemorhina (palpalis group) transmit to livestock trypanosomosis which results in severe losses in affected areas. Little is known about tsetse intraspecific variability and its consequences on the epidemiology of trypanosomosis, justifying further research. Three microsatellite sequences were isolated, cloned and sequenced from a CIRAD/IRD insectariurn sample of Glossina palpalis gambiensis. PCR amplification using primers derived from these microsatellite sequences showed size polymorphisms and mendelian inheritance. These loci were subsequently used for genetic studies in natural populations. At the scale of the geographic distribution of G. palpalis gambiensis, flies originating from Senegal showed genetic differences with flies from Burkina Faso at two of these loci. On the same flies, significant morphometric differences were also found on the wings of the tsetse, measured by a semi -automatical package: Fly Picture Measurement. At a smaller scale, population differenciation was investigated in a cattle-breeding area in Burkina Faso, as part of a multidisciplinary program which aimed at identifying areas at high risk of transmission using GIS. Along the main hydrographic network of the agropastoral zone, flies appeared to be genetically structured within a few kilometers scale. Moreover, in a particular woodland area, microsatellite polymorphism revealed a mixture of genetically distinct sympatric populations. The results leaded to the hypothesis that the origin of the hydrographic network could act on the structuration of these populations of this riverine species of tsetse. This first investigation of intraspecific variability in of tsetse populations using microsatellite DNA polymorphism is discussed in relation to the epidemiology and the control of Africant trypanosornoses. The methodology used could be extended to other vector-borne diseases.