The sea cucumber Holothuria grisea has become the subject of intense and unregulated fishing in northeastern Brazil due to their growing demand in Asian market. However, there is little knowledge about the dynamics and genetics of H. grisea wild populations on the South American coast. In this study, we present the first set of H. grisea microsatellite markers, identified and characterized using Illumina paired-end reads of whole genome shotgun sequencing. From 50 strictlyselected candidates, eight novel microsatellite markers were successfully developed. We then genotyped 30 individuals to evaluate the degree of polymorphism and validate the markers. The number of alleles ranged from three to 14, while observed and expected heterozygotes ranged from 0.156 to 0.906 and from 0.283 to 0.774, respectively. After correcting for multiple tests,we found no evidence of linkage disequilibrium in all pairwise combinations between the loci. One locus (Hgr15607) revealed deviation from the Hardy–Weinberg equilibrium, as well as the presence of null alleles. However, we observed significant differences in frequency distribution between males and females at locus Hgr15607. We believe that the markers describedhere will be useful for conservation efforts and management of H. grisea fisheries and for prospective aquaculture of these organisms.