The cacao tree (Theobroma cacao L.) is native to the Amazon basin and widely cultivated in the tropics to produce seeds, the valuable raw material for the chocolate industry. Conservation of cacao genetic resources and their availability for breeding and production programs are vital for securing cacao supply. However, relatively little is still known about the phylogeographic structure of natural cacao populations. We studied the geographic distribution of cpDNA variation in different populations representing natural cacao stands, cacao farms in Ecuador, and breeding populations. We used six earlier published cacao chloroplast microsatellite markers to genotype 233 cacao samples. In total, 23 chloroplast haplotypes were identified. The highest variation of haplotypes was observed in western Amazonia including geographically restricted haplotypes. Two observed haplotypes were widespread across the Amazon basin suggesting long distance seed dispersal from west to east in Amazonia. Most cacao genetic groups identified earlier using nuclear SSRs are associated with specific chloroplast haplotypes. A single haplotype was common in selections representing cacao plantations in west Ecuador and reference Trinitario accessions. Our results can be used to determine the chloroplast diversity of accessions and in combination with phenotypic assessments can help to select geographically distinctive varieties for cacao breeding programs.