The geographic distribution of genetic variation reflects trends in past population migrations and can be used to make inferences about these migrations. It has been proposed that the east–west orientation of the Eurasian landmass facilitated the rapid spread of ancient technological innovations across Eurasia, while the north–south orientation of the Americas led to a slower diffusion of technology there. If the diffusion of technology was accompanied by gene flow, then this hypothesis predicts that genetic differentiation in the Americas along lines of longitude will be greater than that in Eurasia along lines of latitude. We use 678 microsatellite loci from 68 indigenous populations in Eurasia and the Americas to investigate the spatial axes that underlie population-genetic variation. We find that genetic differentiation increases more rapidly along lines of longitude in the Americas than along lines of latitude in Eurasia. Distance along lines of latitude explains a sizeable portion of genetic distance in Eurasia, whereas distance along lines of longitude does not explain a large proportion of Eurasian genetic variation. Genetic differentiation in the Americas occurs along both latitudinal and longitudinal axes and has a greater magnitude than corresponding differentiation in Eurasia, even when adjusting for the lower level of genetic variation in the American populations. These results support the view that continental orientation has influenced migration patterns and has played an important role in determining both the structure of human genetic variation and the distribution and spread of cultural traits. Am J Phys Anthropol 2011. © 2011 Wiley Periodicals, Inc.