This study examined the population genetic structure of the major malaria vector, Anopheles arabiensis mosquitoes, in Ethiopia and Eritrea. Ethiopia and Eritrea have great geographical diversity, with high mountains, rugged plateaus, deep gorges, and rolling plains. The plateau is bisected diagonally by the Great Rift Valley into the Northwestern Highlands and the Southeastern Highlands. Five A. arabiensis populations from the Northwestern Highlands region and two populations from high-altitude sites in the Great Rift Valley were genotyped using six microsatellite markers to estimate the genetic diversity and population genetic structure of A. arabiensis. We found that A. arabiensis populations from the Northwestern Highlands and the Great Rift Valley region showed a similar level of genetic diversity. The genetic differentiation (F(ST)) of the five mosquito populations within the Northwestern Highlands region was 0.038 (P <.001), while the two populations within the Great Rift Valley showed little genetic differentiation (F(ST) = 0.007, P <.01). The degree of genetic differentiation between the Northwestern Highlands region and the Great Rift Valley region was small but statistically significant (F(ST) = 0.017, P <.001). The population genetic structure of A. arabiensis in the study area did not follow the isolation-by-distance model (r(2) = 0.014, P >.05). The low F(ST) estimates for A. arabiensis populations in Ethiopia and Eritrea are consistent with the general population genetic structure of this species in East Africa based on other molecular markers.