We describe the isolation and development of 13 polymorphic microsatellite loci for the reef-associated dogtooth tuna Gymnosarda unicolor (Perciformes: Scombridae). The loci were tested on 20 individuals from a single population at Flinders Reef, located off the east coast of Australia. No evidence of linkage disequilibrium was detected between any pairs of loci. However, one locus showed a significant departure from Hardy–Weinberg equilibrium. The number of alleles per locus ranged from 2 to 12.