Polytene chromosome studies were undertaken to elucidate taxonomic relationships among populations of Anopheles aquasalis and A. emilianus in Venezuela. Four collection sites were chosen: two in Sucre state (Santa Fe and Guayana) where A. aquasalis (considered to be A. emilianus by Gabaldón and Escalante) is presumed to be the major regional vector of Plasmodium vivax; and two in areas where no malaria transmission occurs (Caño Rico, Aragua state, and Puerto Cabello, Carabobo state). The chromosome banding pattern of the four populations was identical and conformed to the standard chromosome map of A. aquasalis from Brazil. These results suggest that the population from Santa Fe and Guayana, considered to be A. emilianus, is conspecific with A. aquasalis. However, its status as a distinct species with a homosequential polytene chromosome banding pattern cannot be ruled out.