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Background: The Barbirostris Complex comprises six formally described species that cannot be differentiated based on morphology alone. Out of these six species, two have been reported as putative malaria vectors, An. campestris and An. wejchoochotei. Five species are present in Thailand, An. barbirostris, An. campestris, An. dissidens, An. saeungae and An. wejchoochotei, while An. vanderwulpi occurs in Indonesia. As these species cannot be accurately differentiated by morphological characters, there is a crucial lack of information on their bionomics and role in the transmission of malaria and filariasis agents. Results: For differentiating the six species, an allele-specific amplification (AS-PCR) based on the second internal transcribed spacer (ITS2) sequence was developed. From 862 mosquitoes in the Barbirostris Complex collected in 23 provinces throughout Thailand, the AS-PCR was able to identify five species and its validation was undertaken on 185 specimens. Conclusions: This multiplex-PCR assay is potentially able to definitely identify all six species of the Barbirostris Complex and was validated on five species present in Thailand. [ABSTRACT FROM AUTHOR]