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Ancient DNA (aDNA) data are reported for two human skeletons buried within the chancel of the 1608-1616 church at the North American colonial settlement of Jamestown, Virginia. The men are suspected kinsmen of the colony's first Governor, Thomas West, 3 Baron De La Warr based on archaeological, osteological, and documentary evidence. Genomic analyses of these men, Sir Ferdinando Wenman and Captain William West, identify a shared mitochondrial haplogroup, H10e, inferring maternal relatedness. In this unusual case, aDNA prompted further historical research that led to the discovery of illegitimacy, an aspect of identity omitted, likely intentionally, from genealogical records.