Species C of the Anopheles quadrimaculatus complex has a patchy distribution and has been found principally near the coast in river and springs systems of northwest Florida (GULF, SR, SFR--designated as C1, and CHOC--designated as C2) and the Ogeechee River (OGE--C2) near Savannah, Georgia. We have documented significant differences in allelic frequencies at 5 loci (malic enzyme, aconitase, aldehyde oxidase, esterase-5 and esterase-6), and genetic substructuring (Fst) between C1 and C2. The GULF and CHOC populations occurred in apparently similar environments, but were separated by about 160 km. Both these populations are separated from OGE by over 160 km (with low to undetectable distribution of species C in the transect regions). The higher genetic similarity of the CHOC with OGE populations indicated either selective pressures or genetic drift or both as being responsible for the differences between C1 and C2 populations.