Microsatellites are widely used to investigate connectivity and parentage in marine organisms. Despite surgeonfish (Acanthuridae) being dominant members of most reef fish assemblages and having an ecological key role in coral reef ecosystems, there is limited information describing the scale at which populations are connected and very few microsatellite markers have been screened. Here, we developed fourteen microsatellite markers for the convict surgeonfish Acanthurus triostegus with the aim to infer its genetic connectivity throughout its distribution range. Genetic diversity and variability was tested over 152 fishes sampled from four locations across the Indo-Pacific: Mayotte (Western Indian Ocean), Papua New Guinea and New Caledonia (Southwestern Pacific Ocean), and Moorea (French Polynesia). Over all locations, the number of alleles per locus varied from 5 to 24 per locus, and expected heterozygosities ranged from 0.468 to 0.941. Significant deviations from Hardy–Weinberg equilibrium were detected for two loci in two to three locations and were attributed to the presence of null alleles. These markers revealed for the first time a strong and significant distinctiveness between Indian Ocean and Pacific Ocean A. triostegus populations. We further conducted cross-species amplification tests in 13 Pacific congener species to investigate the possible use of these microsatellites in other Acanthuridae species. The phylogenetic placement of A. triostegus branching off from the clade containing nearly all Acanthurus + Ctenochaetus species likely explain the rather good transferability of these microsatellite markers towards other Acanthuridae species. This suggests that this fourteen new microsatellite loci will be helpful tools not only for inferring population structure of various surgeonfish but also to clarify systematic relationships among Acanthuridae.