Anopheles barbirostris, a member of the Barbirostris Subgroup in the Anopheles genus, comprises a complex of species in South and Southeast Asia. This An. barbirostris complex includes eight species such as An. barbirostris sensu stricto (s.s.), An. campestris, An. dissidens, An. donaldi, An. saeungae, An. sarpangensis, An. vanderwulpi, and An. wejchoochotei. This study employed molecular markers, including ITS2 and COI genes, to investigate the phylogenetic relationships within An. barbirostris sensu lato (s.l.) populations from various locations in Indonesia. The analysis reveals the presence of nine distinct populations within this complex, including the first report of An. wejchoochotei in North Sulawesi and a unique Barbirostris population in Magelang, Central Java. This makes it a more complex metapopulation than previously thought. These findings provide critical insights into the diversity of malaria and lymphatic filariasis vectors in Indonesia. Understanding the complex structure of the An. barbirostris populations and their genetic diversity will be useful for effective vector control and disease elimination strategies.