The phylogeographic structure of coral-associated reef fishes may have been severely affected, more than species from deeper habitats, by habitat loss during periods of low sea level. The humbug damselfish, Dascyllus aruanus, is widely distributed across the Indo-West Pacific, and exclusively inhabits branching corals. We used mitochondrial cytochrome b sequence and seven microsatellite loci on D. aruanus samples (260 individuals) from 13 locations across the Indo-West Pacific to investigate its phylogeographic structure distribution-wide. A major genetic partition was found between the Indian and Pacific Ocean populations, which we interpret as the result of geographic isolation on either side of the Indo-Pacific barrier during glacial periods. The peripheral populations of the Red Sea and the Society Islands exhibited lower genetic diversity than, and substantial genetic differences with the other populations, suggesting relative isolation. Thus, vicariance on either side of the Indo-Pacific barrier and peripheral differentiation are thought to be the main drivers that have shaped the phylogeographic patterns presently observed in D. aruanus.