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Historically, the Assiniboine are thought to have split from the Yanktonai Sioux some time in the early 17th century, but this view has been challenged by some linguists, archeologists, and skeletal biologists. Our purpose here is to examine the population structure of the Sioux and the Assiniboine, as reflected in 6 head and 6 body anthropometric dimensions, and to investigate the hypothesis that the Assiniboine diverged relatively recently from the Yanktonai Sioux. For both males and females there is an overall significant division effect, and the FST value indicates a fair amount of differentiation among these closely related groups. The Assiniboine are clearly distinct from all 3 Sioux divisions. The Assiniboine also exhibit a higher within-group phenotypic variance than expected, indicating that their differentiation is due to time and gene flow from outside groups. Among the Sioux divisions the Santee and Yankton-Yanktonai are the most similar, especially in head and face dimensions. The results of this study do not support the historical account of Assiniboine origins. The high degree of differentiation between the Yanktonai and Assiniboine suggests a much more distant split between the Assiniboine and the Sioux than has been traditionally put forth.