The Nassau grouper (Epinephelus striatus), a severely overexploited and endangered coral reef fish in the Western Atlantic, is in urgent need of conservation and management measures. We report the development and characterization of 15 polymorphic, mostly tetranucleotide microsatellite loci based on 40 Nassau grouper samples collected from two Caribbean spawning aggregations. The number of alleles per locus and expected heterozygosities ranged from 4 to 19 and 0.40 to 0.92, respectively. We also demonstrate the cross-amplification utility of these microsatellites for genetic studies of five other grouper species of conservation interest.