Although Spanish chroniclers referred frequently to coerced migration in the Inca Empire, these migrations have been difficult to document archaeologically. One approach to migration studies, strontium isotope (87Sr/86Sr) analysis, has emerged as an effective technique. Until now, however, this method has not been applied to the Inca heartland region of Cuzco, Peru. In this study, we use strontium isotope analysis to examine patterns of prehistoric migration in the Cuzco Valley. Human dental enamel samples from the Cuzco Valley site of Chokepukio are analyzed and compared to the local 87Sr/86Sr signature established through faunal specimens. Though tentative due to a small sample size, the isotope results do not provide evidence for migration at this site from the time periods preceding the rise of the Inca Empire (200 B.C. to A.D. 1400). In contrast, there is substantial evidence for migration during the time of Inca imperialism (A.D. 1400–1532). Among these migrants, variation in 87Sr/86Sr values suggests that individuals emigrated from geologically diverse locations, while sex differences in the migrant group include a higher percentage of females and a greater diversity in female 87Sr/86Sr values. These data, along with ethnohistoric evidence, reveal how Inca labor policies reconfigured the composition of populations in the imperial heartland.