Anopheles pseudopunctipennis is a New World species extending from about 30°S in central Argentina to about 40°N in the USA. It is considered one of the most important malaria vectors in South America and its implication in malaria transmission varies along its distribution. We evaluated the effect of ecoregion and geographical distances on the traits or morphological characters of An. pseudopunctipennis females from 15 localities in Argentina and Bolivia using cladistic and morphometric analyses. The cladistic analysis (Maximum Parsimony) demonstrated that the effect of ecoregion characterization in distinguishing between Bolivian and Argentinean populations was stronger than the effect of geographical distances. Morphometric analysis (Discriminant analysis) did not reveal a pattern between Bolivian and Argentinean populations based on geographical distances. Ecoregional characterization of An. pseudopunctipennis populations throughout is range, caused us to conclude that studies of morphological traits characteristics are necessary in differentiating between populations. Moreover, molecular studies are required to determine whether geographical distances influence population differentiation in An. pseudopunctipennis from Argentina and Bolivia, in the extreme south of South America.