A polymerase chain reaction assay based on differences in the internal transcribed spacer regions of ribosomal DNA was developed for distinguishing 2 members of the Anopheles dirus sibling species complex. This assay distinguished An. dirus species A from species D by producing diagnostic bands, 374 base pairs (bp) in length for species A and 663 bp in length for species D. Both laboratory colonies and field collections from Hainan and Yunnan provinces of China were identified with 100% accuracy.