Tsetse flies of the palpalis group are major vectors of Human African Trypanosomiasis in Africa. Accurate knowledge of species identity is essential for vector control. Here, we combine ribosomal internal transcribed spacer 1 (ITS1), mitochondrial Cytochrome Oxidase 1 (COI) and microsatellites to determine the population structure and phylogenetic relations of Glossina p. palpalis in Equatorial Guinea. CO1 sequence data suggest that G. p. palpalis in Equatorial Guinea is a distinct subspecies from previously described G. p. palpalis in West Africa and Democratic Republic of Congo. Glossina p. palpalis in Equatorial Guinea and DRC share a common ancestor which diverged from West African G. p. palpalis around 1.9 Ma. Previous ITS1 length polymorphism data suggested the possible presence of hybrids in Equatorial Guinea. However, ITS1 showed incomplete lineage sorting compared with clearly defined COI groups, and data from 12 unlinked microsatellites provided no evidence of hybridization. Microsatellite data indicated moderate but significant differentiation between the populations analysed (Rio Campo, Mbini and Kogo). Moreover, unlike previous studies of G. p. palpalis, there was no evidence for heterozygote deficiency, presence of migrants or cryptic population structure. Variance effective population size at Rio Campo was estimated at 501–731 assuming eight generations per year. This study of the population genetics of G. p. palpalis in central Africa provides the first estimate of genetic differentiation between geographically separated G. p. palpalis populations.