Genetic distance measurements are an important tool to differentiate field populations of disease vectors such as the mosquito vectors of malaria. Here, we have measured the genetic differentiation between Anopheles arabiensis and Anopheles gambiae, as well as between proposed emerging species of the latter taxon, in whole genome scans by using 23-25 microsatellite loci. In doing so, we have reviewed and evaluated the advantages and disadvantages of standard parameters of genetic distance, F ST, RST, (␦ ) 2 , and D. Further, we have introduced new parameters, D and D K, which have well defined statistical significance tests and complement the standard parameters to advantage. D is a modification of D, whereas D K is a measure of covariance based on Pearson's correlation coefficient. We find that A. gambiae and A. arabiensis are closely related at most autosomal loci but appear to be distantly related on the basis of X-linked chromosomal loci within the chromosomal Xag inversion. The M and S molecular forms of A. gambiae are practically indistinguishable but differ significantly at two microsatellite loci from the proximal region of the X, outside the Xag inversion. At one of these loci, both M and S molecular forms differ significantly from A. arabiensis, but remarkably, at the other locus, A. arabiensis is indistinguishable from the M molecular form of A. gambiae. These data support the recent proposal of genetically differentiated M and S molecular forms of A. gambiae.M any major infectious diseases, such as malaria, leishmaniasis, and sleeping sickness, are transmitted by insect vectors. Molecular genetic markers have become powerful tools for elucidating the population biology and evolution of such vectors, topics that are highly relevant to disease transmission in the field (1-4). Genetic variation in vector populations contributes to their susceptibility to infection by the pathogen, their degree of anthropophily, their daily survival and reproductive rates, and the epidemiology of the disease in the human host (5). A case in point is the African mosquito of the Anopheles gambiae (sensu latu) complex (5). These include the most important vector of human malaria, A. gambiae (sensu strictu), as well as closely related species that are significant vectors in specific areas (e.g., Anopheles arabiensis) or are altogether unable to serve as vectors (Anopheles quadriannulatus). Furthermore, even within A. gambiae s.s., cytologically defined chromosomal forms (e.g., Mopti, Savanna, and Bamako) are reproductively isolated in the northern dry areas of West Africa, including Mali and Burkina Faso, and may represent emerging species with different disease transmission characteristics (5, 6). Although many DNA regions have been recently analyzed to examine genetic differentiation within A. gambiae s.s, the only fixed molecular differences found so far that consistently discriminate chromosomal forms are in the X-linked ribosomal (r)DNA region (1-4, 7). In Mali and Burkina Faso, these markers distinguish Mopti from Sava...