The population genetic structure of the African malaria vector Anopheles arabiensis from western Kenya, the Great Rift Valley, and coastal Kenya was investigated using 12 microsatellite loci and a partial sequence of mtDNA dehydrogenase gene subunit 5 (ND5). The mean number of alleles and the observed heterozygosity were similar for the mosquito populations from the three regions as revealed by the microsatellite data. A total of 30 polymorphic sites in the ND5 gene defined 39 haplotypes. Six haplotypes were shared among four populations from the three distinct ecological conditions, and they constituted 92% of the total number of individuals sequenced. Mitochondrial haplotype and nucleotide diversity were high. Microsatellite markers within polymorphic inversions revealed a level of genetic differentiation (F-ST = 0.116) four to seven times higher than markers outside inversions (F-ST = 0.016) or inside fixed inversions (F-ST = 0.027). Mitochondrial ND5 gene sequences did not reveal significant genetic differentiation for the same four populations (Phi(ST) = -0.008). The contrasts in the level of genetic differentiation between microsatellite markers inside polymorphic inversions, the mitochondrial ND5 gene, and microsatellite markers outside inversions suggest that the level of genetic differentiation in An. arabiensis populations across the Great Rift Valley varies significantly among different areas of the genome. Variations in the degree of genetic differentiation with respect to the chromosomal location of microsatellite markers may result from intrinsic characteristics of the markers, demographic or historic factors affecting these populations, and the possible adaptive significance of chromosomal inversions to climatic conditions.