Although many reef fishes of the tropical Atlantic are widely distributed, there are large discontinuities that may strongly influence phylogeographical patterns. The freshwater outflow of the Amazon basin is recognized as a major barrier that produces a break between Brazilian and Caribbean faunas. The vast oceanic distances between Brazil and the mid-Atlantic ridge islands represent another formidable barrier. To assess the relative importance of these barriers, we compared a fragment of the mitochondrial DNA (mtDNA) cytochrome b gene among populations of three species of Atlantic surgeonfishes: Acanthurus bahianus, A. chirurgus and A. coeruleus. These species have similar life histories but different adult habitat preferences. The mtDNA data show no population structure between Brazil and the mid-Atlantic islands, indicating that this oceanic barrier is readily traversed by the pelagic larval stage of all three surgeonfishes, which spend ~45–70 days in the pelagic environment. The Amazon is a strong barrier to dispersal of A. bahianus (d = 0.024, ΦST = 0.724), a modest barrier for A. coeruleus (ΦST = 0.356), and has no discernible effect as a barrier for A. chirurgus. The later species has been collected on soft bottoms with sponge habitats under the Amazon outflow, indicating that relaxed adult habitat requirements enable it to readily cross that barrier. A limited ability to use soft bottom habitats may also explain the low (but significant) population structure in A. coeruleus. In contrast, A. bahianus has not been collected over deep sponge bottoms, and rarely settles outside shallow reefs. Overall, adult habitat preferences seem to be the factor that differentiates phylogeographical patterns in these reef-associated species.