The pathogenic agent of human African trypanosomiasis (HAT) is a trypanosome belonging to the species Trypanosoma brucei s.l. Molecular methods developed for typing T. brucei s.l. stocks are for the most part not polymorphic enough to study genetic diversity within T. brucei gambiense (T. b. gambiense) group 1, the main agent of HAT in West and Central Africa. Furthermore, these methods require high quantities of parasite material and consequently are hampered by a selection bias of the isolation and cultivation techniques. In this study, we evaluated the potential value of microsatellite markers (eight loci) in the genetic characterisation of T. brucei s.l. compared to the multi-locus enzyme electrophoresis reference technique. Stocks isolated in Ivory Coast and reference stocks were used for this purpose. Microsatellite markers were shown to be polymorphic enough to evidence the existence of genetic diversity within T. b. gambiense group 1 and to show the existence of mixed infections. Furthermore, they were able to amplify trypanosome DNA directly from field samples without the usual culturing stages. While the ability of microsatellite markers to detect mixed infections in such field samples is currently being discussed, they appear to be useful to study the parasite population's geographical structure and may provide new insight into their reproductive mode, a topic that is still under debate. Thus, use of microsatellite markers will contribute to the study of the influence of parasite genetics in the diversity of responses to HAT and may contribute to the improvement of HAT molecular diagnosis.