The common practice of resettlement and the development of administrative and ceremonial systems shaped the population landscape of the Andean region under the Inca rule. The area surrounding Coropuna and Solimana volcanoes, in the Arequipa region (Peru), carried a high‐density, multiethnic population. We studied the genetic variation among three pre‐Columbian populations from three functionally diverse archaeological sites excavated in this region. By analyzing the genetic composition of a large ceremonial center (Acchaymarca), an isolated pastoral settlement (Tompullo 2), and an agricultural settlement characterized by architectural features rare in the region (Puca), we investigated the patterns of population movements and the distribution of genetic diversity. We obtained mitochondrial DNA sequences for 25 individuals and autosomal microsatellite profiles for 20 individuals from Acchaymarca and Puca sites. These were compared with previously published genetic data for Tompullo 2 and other pre‐Columbian populations. We found differences among the genetic portraits of the three populations, congruent with the archaeologically described functions and characteristics of the sites. The Acchaymarca population had the highest genetic diversity and possessed the lowest number of unique mtDNA haplotypes. The Tompullo 2 population exhibited the lowest level of genetic diversity. The Puca population was distinct from the other two populations owing to a high frequency of haplogroup A haplotypes, what potentially explains the non‐local character of the burial architecture. Our analyses of microsatellite data suggest that gene flow between sites was mostly mediated by females, which is consistent with ethnohistorical knowledge of the social organization of the pre‐Columbian communities. Am J Phys Anthropol, 2014. © 2014 Wiley Periodicals, Inc.