The butterfly Troides aeacus formosanus (Rothschild) (Lepidoptera: Papilionidae) is one of the five T. aeacus subspecies adapted to tropical regions and is endemic to Taiwan. Partial DNA sequences of cytochrome oxidase I (COI) and amplified fragment length polymorphism (AFLP) markers were analyzed to differentiate this birdwing butterfly subspecies and to indicate its possible dispersal history. Phylogenetic relationship analyses have shown that T. a. formosanus can be divided into lineages I and II with limited COI variations, and AFLP patterns also have shown two divergent clusters, A and B. In populations collected from southern Taiwan, there was a significant relationship between lineage II and cluster B. Parsimonious network of COI indicated haplotype H1, the nearest to the other subspecies, should be the basal form in T. a. formosanus. Both COI sequences and AFLP patterns indicated that haplotype diversity was high in all populations, whereas low levels of genetic differentiation were found among populations. Molecular variance suggested that most of the total variance was due to differences within populations. These results, together with the divergence estimation and the specific geological topology in southern Taiwan, indicate that T. a. formosanus might have entered Taiwan during recent glaciations (≈150,000 yr ago) and become adapted to the tropical environment. The observed differentiation of T. a. formosanus could have resulted from the geographic barriers of the Central Range on the island. Moreover, genetic exchanges caused by movement of individuals among butterfly farms or gardens in the past two decades may have led to less diversification among current populations of T. a. formosanus.