This study couples biological data with aspects of material culture and mortuary ritual for several sites within the proposed Coosa chiefdom described by sixteenth-century Spanish accounts to explore how cultural identities were actively constructed and maintained within the region. The primary goal is to examine regional interactions between these communities and their constructions of social identity and sociopolitical dynamics vis à vis their biological affinities. Questions regarding regional interactions between these groups have been a stimulus for archaeological debate. These interactions may have played a crucial role in the construction of separate cultural identities. What is not clear is to what extent differences in cultural identity reflect or are related to differences in biological relationships. The skeletal samples used in this study represent six Late Mississippian archaeological sites assigned to three archaeological phases: the Dallas Phase, Fains Island (40JE1), Cox (40AN19), and David Davis (40HA301) sites; the Mouse Creek Phase, Ledford Island (40BY13) site; and the Barnett Phase, King (9FL5) and Little Egypt (9MU102) sites. Twenty-seven dental and 22 cranial nonmetric traits were recorded for 923 individuals. Biological affinities were calculated using the Mahalanobis D2 statistic for the cranial and dental non-metric traits. Biological Distance measures were compared to a geographic matrix to examine isolation by distance between the sites. Further analysis was conducted by constructing an R matrix to examine levels of heterogeneity. Comparisons between biological distance and geographical distances suggest that the samples used in this analysis do not conform to the expected isolation-by-distance model. Furthermore, East Tennessee groups appear distinct from their North Georgia neighbors suggesting little biological interaction between these groups. The results of the biological distance analysis conforms to differences in material culture and mortuary ritual between these groups. The results suggests that if there was a political alliance within the region for this period it is not associated with biological relatedness nor did it act as a unifying force for individual communities’ cultural identity.