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Background: Although Sri Lanka is considered as a malaria-free nation, the threat of re-emergence of outbreaks still remains due to the high prevalence and abundance of malaria vectors. Analysis of population genetic structure of malaria vectors is considered to be one of the vital components in implementing successful vector control programmes. The present study was conducted to determine the population genetic structure of three abundant malaria vectors; Anopheles subpictus sensu lato (s.l.) , Anopheles peditaneatus and Anopheles vagus from five administrative districts in two climatic zones; intermediate zone (Badulla and Kurunegala districts) and dry zone (Ampara, Batticoloa and Jaffna districts) of Sri Lanka using the mitochondrial gene, cytochrome c oxidase subunit I ( COI ). Methods: Adult mosquitoes of An. subpictus s.l. , An. peditaeniatus, and An. vagus were collected from five study sites located in five districts using cattle baited traps and backpack aspirators. Representative samples of each species that were morphologically confirmed were selected from each locality in generating COI sequences (> 6 good quality sequences per species per locality). Results: Anopheles subpictus s.l. specimens collected during the study belonged to two sibling species; An. subpictus 'A' (from all study sites except from Jaffna) and An. subpictus 'B' (only from Jaffna). The results of haplotype and nucleotide diversity indices showed that all the three species are having high genetic diversity. Although a high significant pairwise difference was observed between An. subpictus ' A' and 'B' ( F st > 0.950, p < 0.05), there were no significant genetic population structures within An. peditaeniatus , An. vagus and An. subpictus species A ( p > 0.05), indicating possible gene flow between these populations. Conclusions: Gene flow among the populations of An. peditaeniatus , An. vagus and An. subpictus species A was evident. Application of vector control measures against all mosquito species must be done with close monitoring since gene flow can assist the spread of insecticide resistance genes over a vast geographical area. [ABSTRACT FROM AUTHOR]