Although the presence of Tiwanaku-style material culture throughout southern Peru, northern Chile, and western Bolivia is well documented, the nature of Tiwanaku influence during the Middle horizon (A.D. 500–1100) is variously attributed to imperial expansion or economic and/or religious relationships. Strontium isotope data from archaeological human remains from Tiwanaku-affiliated sites identified first-generation immigrants from the Lake Titicaca basin outside of the Tiwanaku heartland at the Peruvian site of Chen Chen. These data provide an important component to studies that demonstrated close biological relationships during the Middle horizon but could not demonstrate the direction of population movement. However, no immigrants from the Lake Titicaca basin were identified at the San Pedro de Atacama cemeteries of Coyo Oriental, Coyo-3, and Solcor-3. At the sites of Tiwanaku, Tilata, Iwawe, and Kirawi, strontium isotope ratios were also variable, and demonstrate movement within the Lake Titicaca basin. This demonstrates that Tiwanaku influence involved direct colonization in the Moquegua Valley but that in other regions, like San Pedro de Atacama, local inhabitants adopted Tiwanaku-style material culture. This elucidates the complex and highly variable relationships between the Tiwanaku heartland and peripheral sites during the Middle horizon.