Abstract We have estimated DNA sequence variation within and between two populations of Drosophila ananassae, using six-cutter restriction site variation at vermilion (v) and furrowed (fw). These two gene regions are located close to the centromere on the left and right X chromosome arms, respectively. In the fw region, no DNA polymorphism was detected within each population. In the v region, average heterozygosity per nucleotide was very low in both populations (pi = 0.0005 in the Burma population, and 0.0009 in the India population). These estimates are significantly lower than those from loci in more distal gene regions. The distribution of DNA polymorphisms between both populations was also striking. At fw, three fixed differences between the Burma and India populations were detected (two restriction site differences and one insertion/deletion of approximately 2 kb). At v, each DNA polymorphism in high frequency in the total sample was nearly fixed in one or the other population, although none of them reached complete fixation. The observed pattern of reduced variation within populations and fixed differences between populations appears to correlate with recombination rate. We conclude that recent hitchhiking associated with directional selection is the best explanation for this pattern. The data indicate that different selective sweeps have occurred in the two populations. The possible role of genetic hitchhiking in rapid population differentiation in gene regions of restricted recombination is discussed.