Abstract The dusky grouper, Epinephelus marginatus, is a well-known and widespread marine fish assessed as endangered by the International Union for the Conservation of Nature. Analyzing the genetic diversity of this species is, therefore, of utmost importance and necessary for conservation purposes. Microsatellites are molecular tools with advantages that are ideal for population analyses. This study provides the first set of species-specific microsatellite loci for E. marginatus that can be applied when assessing both intra- and interpopulation genetic variation. Twenty microsatellite loci were isolated and characterized for the dusky grouper by genotyping 20 individuals obtained from the North Eastern Atlantic Ocean (n = 4) and from the South Western Atlantic Ocean (n = 16). The number of alleles per locus varied from 2 to 11, while the observed and expected heterozygosities ranged from 0.25 to 0.94 and 0.34 to 0.89, respectively. The polymorphic information content varied from moderately to highly informative. This suite of markers provides the first specific nuclear tools for E. marginatus and, thus, allows to assess with more specificity different populations’ structures.