The brown-throated three-toed sloth (Bradypus variegatus) is an extremely sedentary arboreal mammal found in many Neotropical forests. Because of its low dispersal potential and ease in capture, it has the potential to serve as a model species to investigate the effects of land cover change in the Neotropics on gene flow and population connectivity. To better understand aspects of B. variegatus biology, such as mating system and dispersal rates, we isolated and characterized 18 polymorphic microsatellite markers. Markers were tested using 32 B. variegatus individuals sampled from a site in northeastern Costa Rica. Each locus contained between three and 12 alleles, while mean expected and observed heterozygosity were equal to 0.72. No loci deviated from Hardy–Weinberg equilibrium, and one locus was significantly linked to two others. These markers have sufficient polymorphism to identify individuals and assign parentage, and can further be used to investigate dispersal rates, mating structure, and other aspects of three-toed sloth ecology.