Malaria continues to be a public health problem of high priority in the majority of malaria endemic countries of the South East Asia region, such as Indonesia. Vector control remain the most effective measure to prevent malaria transmission. The study of mosquito species complexes and the understanding of the biology, evolution as well as epidemiology of malaria transmission and the possibly of development of efficient control measures against malaria have been subjects of greatly increased interest in recent years. Natural populations of Anopheles barbirostris Van der Wulp were sampled from 5 geographically isolated populations in Indonesia: (1) Ambarawa, Central Java. (2) Tara-Tara, North Sulawesi, (3) Boru-Boru, (4) Konga and (5) Tanjung Bunga, the last three in Flores, East Nusa Tenggara. Analysis of larval mitotic chromosomes had been made from F I progeny of isofemale lines derived from these 5 populations. Specimens from these populations appear to share a similar mitotic karyotype (2n=6) consisting of two pairs of autosomes and one pair of sex chromosomes. The Giemsa staining of mitotic chromosomes of the An. barbirostris complex showed considerable variation in the amount and distribution of constitutive heterochromatin in sex chromosomes. These cytological differences have led to the recognation of 4 distinct cytological forms within the taxon An. barbirostris, viz., form A (X1,X2,X3,Y1), form B (X1,X2,X3,Y2) form C (X2,X3,Y3) and form D (X2,X3,Y4). Form A is widely distributed in Indonesia, while, form Band D have been found in sympatric association with form A in Tara-tara 2, North Sulawesi; Konga, Flores and Tanjung Bunga, Form C, however, has been found only in Boru-Boru, and sympatric with forms A and B.