Criollo cacao (Theobroma cacao ssp. cacao) was cultivated by the Mayas over 1500 years ago. It has been suggested that Criollo cacao originated in Central America and that it evolved independently from the cacao populations in the Amazon basin. Cacao populations from the Amazon basin are included in the second morphogeographic group: Forastero, and assigned to T. cacao ssp. sphaerocarpum. To gain further insight into the origin and genetic basis of Criollo cacao from Central America, RFLP and microsatellite analyses were performed on a sample that avoided mixing pure Criollo individuals with individuals classified as Criollo but which might have been introgressed with Forastero genes. We distinguished these two types of individuals as Ancient and Modern Criollo. In contrast to previous studies, Ancient Criollo individuals formerly classified as 'wild', were found to form a closely related group together with Ancient Criollo individuals from South America. The Ancient Criollo trees were also closer to Colombian-Ecuadorian Forastero individuals than these Colombian-Ecuadorian trees were to other South American Forastero individuals. RFLP and microsatellite analyses revealed a high level of homozygosity and significantly low genetic diversity within the Ancient Criollo group. The results suggest that the Ancient Criollo individuals represent the original Criollo group. The results also implies that this group does not represent a separate subspecies and that it probably originated from a few individuals in South America that may have been spread by man within Central America.