Anopheles arabiensis Patton is one of the principal vectors of malaria in sub-Saharan Africa, occupying a wide variety of ecological zones. This species is increasingly responsible for malaria transmission in Africa and is becoming the dominant vector species in some localities. Despite its growing importance, little is known about genetic polymorphisms in this species. Multiple sequences of various gene fragments from An. arabiensis isolates from Cameroon were obtained from GenBank. In total, 20 gene fragments containing single-nucleotide polymorphisms (SNPs) at moderate density were selected for direct sequencing from field collected specimens from Tanzania and Zambia. We obtained 301 SNPs in total from the 20 gene fragments, 60 of which were suitable for Illumina GoldenGate SNP genotyping. A greater number of SNPs (n = 185) was suitable for analysis using Sequenom iPLEX, an alternative high-throughput genotyping technology using mass spectrometry. An SNP was present every 59 (+/- 44.5) bases on average. Overall, An. arabiensis from Tanzania and Zambia are genetically closer (mean F(ST) = 0.075) than either is to populations in Cameroon (F(ST, TZ-CM) = 0.250, F(ST,ZA-CM) = 0.372). A fixed polymorphism between East/southern and Central Africa was identified on AGAP000574, a gene on the X chromosome. We have identified SNPs in natural populations of An. arabiensis. SNP densities in An. arabiensis were higher than Anopheles gambiae s.s., suggesting a greater challenge in the development of high-throughput SNP analysis for this species. The SNP markers provided in this study are suitable for a high-throughput genotyping analysis and can be used for population genetic studies and association mapping efforts.