The prickly redfish Thelenota ananas (Jaeger, 1833) is a widely distributed tropical sea cucumber. Populations of this species have been increasingly harvested throughout its distribution area in the Indo-Pacific region, which led to significant overexploitation issues. In order to investigate the genetic structure, diversity and connectivity of its populations, 42 microsatellite loci were isolated from a T. ananas microsatellite-enriched DNA library. These loci were characterized on 24 individuals collected from the Seychelles. The number of alleles per locus ranged from 3 to 22. The observed heterozygosity ranged from 0.083 to 0.952, while the expected heterozygosity ranged from 0.162 to 0.974. No linkage disequilibrium was detected among all loci and 20 loci (48%) were at Hardy–Weinberg equilibrium. These 42 loci represent useful tools for assessing genetic diversity, structure and gene flow among T. ananas populations, providing relevant knowledge for the management and conservation of those major commercial resources.