Endemic species at remote islands have a high risk of extinction because they often exhibit ecological, biological and genetic traits that make them particularly vulnerable to disturbances. McCulloch's anemonefish (Amphiprion mccullochi) is endemic to a few oceanic reefs off Australia's east coast and is an habitat specialist. Using 454 shotgun sequencing, we developed and evaluated primers for seventeen independent microsatellite loci to reveal gene flow, population genetic structure and genetic diversity across three isolated reefs. Observed and expected heterozygosities ranged from 0.556 to 1 and 0.543 to 0.907, respectively, in 30 Lord Howe Island individuals. When cross tested with the close relative, Amphiprion akindynos, amplification was successful with high levels of polymorphism. These loci will therefore be useful in studies of A. mccullochi, A. akindynos and possibly other closely related anemonefish.