Alternative models have been proposed to explain the formation and decline of the south Peruvian Nasca culture, ranging from migration or invasion to autochthonous development and ecological crisis. To reveal to what extent population dynamic processes accounted for cultural development in the Nasca mainland, or were influenced by them, we analyzed ancient mitochondrial DNA of 218 individuals, originating from chronologically successive archaeological sites in the Palpa region, the Paracas Peninsula, and the Andean highlands in southern Peru. The sampling strategy allowed a diachronic analysis in a time frame from approximately 800 BC to 800 AD. Mitochondrial coding region polymorphisms were successfully analyzed and replicated for 130 individuals and control region sequences (np 16021–16408) for 104 individuals to determine Native American mitochondrial DNA haplogroups and haplotypes. The results were compared with ancient and contemporary Peruvian populations to reveal genetic relations of the archaeological samples. Frequency data and statistics show clear proximity of the Nasca populations to the populations of the preceding Paracas culture from Palpa and the Peninsula, and suggest, along with archaeological data, that the Nasca culture developed autochthonously in the Rio Grande drainage. Furthermore, the influence of changes in socioeconomic complexity in the Palpa area on the genetic diversity of the local population could be observed. In all, a strong genetic affinity between pre-Columbian coastal populations from southern Peru could be determined, together with a significant differentiation from ancient highland and all present-day Peruvian reference populations, best shown in the differential distribution of mitochondrial haplogroups. Am J Phys Anthropol 2010. © 2009 Wiley-Liss, Inc.