Abstract: Annatto (Bixa orellana) is a tropical crop native to the Americas with Amazonia as the likely center of origin of domestication. Annatto is important because it produces the dye bixin, which is widely used in the pharmaceutical, cosmetic and textile industries. A total of 32 microsatellite loci were iso lated from a microsatellite-enriched genomic library, of which 12 polymorphic loci were used to characterize four populations of B. orellana and B. orellana var. urucurana, the wild relative. Higher genetic diversity estimates were detected for the wild populations when compared to the cultivated populations. Also, higher apparent utcrossing rates were found for the two wild than the cultivated populations. These results indicate a mixed mating system for the species. All markers described herein have potential to be used in further studies evaluating the genetic diversity, population dynamics, domestication, breeding, and conservation genetics of annatto.