We describe the isolation, characterization, and application of 11 polymorphic microsatellite loci from the tropical sea urchin Tripneustes gratilla. We genotyped a total of 73 individuals in three subpopulations (two in Hawaii, and one in the Philippines) and found between eight and 33 alleles per locus. We found significant heterozygote deficits within at least one subpopulation at seven out of 11 loci. We were successful at cross-amplifying four loci in the congeneric Tripneustes ventricosus. When used appropriately, this set of loci will be useful for quantifying population structure in Tripneustes urchins across broad geographical ranges.