Ancient DNA was recovered from 17 individuals found in a rock shelter in the district of "La Purnia" (Santander, Colombia). This region is the homeland of pre-Columbian Guane, whom spread over the "Río Suarez" to the "Río de Oro", and were surrounded to the west by the Central Andes, south and east by foothills of Eastern Andes, and north by the "Chicamocha" river canyon. Guanes established in a region that straddles the Andes and the northern Amazon basin, possibly making it an unavoidable conduit for people moving to and from South America. We amplified mtDNA hypervariable region I (HVI) segments from ancient bone remains, and the resulting sequences were compared with both ancient and modern mitochondrial haplogroups from American and non-American populations. Samples showed a distribution of 35% for haplogroup A, 41% for haplogroup B and 24% for haplogroup D. Nine haplotypes were found in 17 samples, indicating an unusually high genetic diversity on a single site ancient population. Among them, three haplotypes have not been previously found in America, two are shared in Asia, and one is a private haplotype. Despite geographical barriers that eventually isolated them, an important influence of gene flow from neighboring pre-Columbian communities, mainly Muiscas, could explain the high genetic polymorphism of this community before the Spanish conquest, and argues against Guanes as being a genetic isolate.