The haplogroup identities of 800 mtDNAs randomly and systematically selected to be representative of the population of Puerto Rico were determined by restriction fragment length polymorphism (RFLP), revealing maternal ancestries in this highly mixed population of 61.3% Amerindian, 27.2% sub-Saharan African, and 11.5% West Eurasian. West Eurasian frequencies were low in all 28 municipalities sampled, and displayed no geographic patterns. Thus, a statistically significant negative correlation was observed between the Amerindian and African frequencies of the municipalities. In addition, a statistically highly significant geographic pattern was observed for Amerindian and African mtDNAs. In a scenario in which Amerindian mtDNAs prevailed on either side of longitude 66°16′ West, Amerindian mtDNAs were more frequent west of longitude 66°16′ West than east of it, and the opposite was true for African mtDNAs. Haplogroup A had the highest frequency among Amerindian samples (52.4%), suggesting its predominance among the native Taínos. Principal component analysis showed that the sub-Saharan African fraction had a strong affinity to West Africans. In addition, the magnitudes of the Senegambian and Gulf of Guinea components in Puerto Rico were between those of Cape Verde and São Tomé. Furthermore, the West Eurasian component did not conform to European haplogroup frequencies. HVR-I sequences of haplogroup U samples revealed a strong North African influence among West Eurasian mtDNAs and a new sub-Saharan African clade. Am J Phys Anthropol 128:131-155, 2005. © 2005 Wiley-Liss, Inc.