Background: Anopheles (Kerteszia) cruzii was the most important vector of human malaria in southern Brazil between 1930–1960. Nowadays it is still considered an important Plasmodium spp. vector in southern and south-eastern Brazil, incriminated for oligosymptomatic malaria. Previous studies based on the analysis of X chromosome banding patterns and inversion frequencies in An. cruzii populations from these areas have suggested the occurrence of three sibling species. In contrast, two genetically distinct groups among An. cruzii populations from south/south-east and north-east Brazil have been revealed by isoenzyme analysis. Therefore, An. cruzii remains unclear.