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Paleolithic archaeological and skeletal remains from the Nile Valley have yielded a complex picture of life along the river. Sociocultural and sociopolitical events during this time frame shaped population structure, while gene flow and genetic drift further developed it. In this study, we took a population genetics approach to modeling Nubian biological relationships in an effort to describe how an accumulation of events formed Nubian population structure. A variety of Nubian samples were utilized, spanning the Mesolithic-Christian time periods and geographically from just above the first through the third cataracts. Population genetics statistics were employed to estimate and depict biological affinities (Mahalanobis D2 with a tetrachoric matrix, principal coordinates analysis, FST, and Relethford-Blangero residuals), supplemented by spatial-temporal modeling (Mantel tests and PROTESTs). Variation was high among these groups, indicating an intricate pattern of relationships in their population history where similar levels of gene flow probably stemmed from extensive cultural contact with Egypt and other populations in a variety of contexts. Genetic drift was also apparent in some of these sites, which is consistent with social and political histories of these groups. Traditional modeling of spatial-temporal patterning was not successful, which may be attributed to the nonlinear, loose clustering of Nubian groups by site. Collectively, the archaeological, biological, and environmental evidence supports the ideas of multiple populations living in Lower Nubia during the Paleolithic and/or a new population entering the area and shaping Nubian population structure.