Annatto (Bixa orellana) is an important colorant domesticated in the Neotropics, although it is not clear where or from which wild populations. We reviewed the available biological, archaeological, and ethnographic information about annatto, and integrated this with our recent ethnobotanical observations of cultivated and non–cultivated populations in order to evaluate the hypothesis that what is classified as Bixa urucurana is the wild ancestor of cultivated annatto, Bixa orellana. Most B. urucurana populations we found in Amazonia occurred in open forests or anthropogenic landscapes, although never cultivated, and always associated with riparian environments. While cultivated annatto always produces abundant pigment, B. urucurana populations that we observed contained variable amounts of pigment, from very little to nearly the amount of cultivated annatto, suggesting gene flow from cultivated to non–cultivated. Bixa urucurana has indehiscent fruits, which indicate changes in dehiscence during annatto domestication, a notable feature rarely found in other tree species. Local residents identified the non–cultivated populations as wild annatto (urucum bravo), and they emphasized their smaller fruits with less pigment, their spontaneous regeneration, their non–use, and that they hybridize with cultivated annatto. Ethnography identified the symbolic importance of annatto, but an explicit mention of origin only comes from southern Amazonia. Although the oldest annatto archaeological record came from the Caribbean, domestication occurred in northern South America, since B. urucurana does not occur in the Caribbean. Traditional ecological knowledge and morphology identified the close relationship between B. urucurana (never cultivated) and B. orellana (always cultivated). Evidence reported here strongly supports Kuntze’s (1925) suggestion that Bixa urucurana Willd. is a variety of B. orellana L., thus identifying the wild ancestor of cultivated annatto.