Indigenous peoples have occupied the island of Puerto Rico since at least 3000 B.C. Due to the demographic shifts that occurred after European contact, the origin(s) of these ancient populations, and their genetic relationship to present-day islanders, are unclear. We use ancient DNA to characterize the population history and genetic legacies of pre-contact Indigenous communities from Puerto Rico. Bone, tooth and dental calculus samples were collected from 124 individuals from three pre-contact archaeological sites: Tibes, Punta Candelero and Paso del Indio. Despite poor DNA preservation, we used target enrichment and high-throughput sequencing to obtain complete mitochondrial genomes (mtDNA) from 45 individuals and autosomal genotypes from two individuals. We found a high proportion of Native American mtDNA haplogroups A2 and C1 in the pre-contact Puerto Rico sample (40% and 44%, respectively). This distribution, as well as the haplotypes represented, support a primarily Amazonian South American origin for these populations, and mirrors the Native American mtDNA diversity patterns found in present-day islanders. Three mtDNA haplotypes from pre-contact Puerto Rico persist among Puerto Ricans and other Caribbean islanders, indicating that present-day populations are reservoirs of pre-contact mtDNA diversity. Lastly, we find similarity in autosomal ancestry patterns between pre-contact individuals from Puerto Rico and the Bahamas, suggesting a shared component of Indigenous Caribbean ancestry with close affinity to South American populations. Our findings contribute to a more complete reconstruction of pre-contact Caribbean population history and explore the role of Indigenous peoples in shaping the biocultural diversity of present-day Puerto Ricans and other Caribbean islanders.