The archaeological site of Santa Ana-La Florida (SALF), located in the Ecuadorian upper Amazon, is in the region of Theobroma spp. greatest genetic diversity, thus making it ideal to investigate the origins of domestication of this enigmatic tree. We present research showing that the residents of SALF were involved in the domestication of cacao, traditionally thought to have been first domesticated in Mesoamerica and/or Central America. We used three independent lines of evidence—starch grains, theobromine residues and ancient DNA—dating from approximately 5,300 years ago, to establish the earliest evidence of T. cacao use in the Americas, the first unequivocal archaeological example of its pre-Columbian use in South America and reveal the upper Amazon region as the oldest centre of cacao domestication yet identified. We suggest that new paleoethnobotanical research will expand our knowledge of this process, including the timing, locations, and uses of cacao by Indigenous South Americans.