There has been an increase in malaria cases in southern African countries in recent years due to the presence of populations of Anopheles funestus that are resistant to the pyrethroid class of insecticides. Since A. funestus is one of the major African malaria vectors, knowledge of its genetic structure will benefit control strategies, such as the management of insecticide resistance, by allowing predictions to be made of possible spread of the resistance. This study uses microsatellite DNA markers to analyze samples from five countries in east (Kenya and Uganda), central (Malawi) and southern (South Africa and Mozambique) Africa. There were deviations from Hardy-Weinberg expectations for some loci in all population samples but this was probably due to the presence of null alleles. High levels of genetic diversity were observed (mean alleles per locus = 6.5-10; unbiased H=0.23-0.89). Low differentiation was observed between Kenya and Uganda (average F(ST)=0.002, R(ST)=0.0001) and between Mozambique and South Africa (F(ST)=0.0004, R(ST)=0.02), contrary to high differentiation among the central and southern Africa samples (average F(ST)=0.023, R(ST)=0.027). High differentiation was measured across the region (mean F(ST)=0.04, R(ST)=0.08), east versus Malawi (F(ST)=0.067, R(ST)=0.089) or southern Africa populations (F(ST)=0.068, R(ST)=0.15). A test of isolation by distance along the east-central-south transect gave evidence (R(2)=0.50, P<0.001) that geographic distance limits gene flow in A. funestus.