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While the British origins of North American colonization currently are widely accepted, there is new evidence that other countries and non-Christians may have been earlier in establishing permanent settlements on the North Atlantic coast. Using the new research tool of human genomics, this paper provides DNA evidence that Croatians and Sephardic Jews were absorbed into the ancestral population of the Lumbee Native American tribe of North Carolina during the mid- to late-1500s. We further propose that these Sephardic Jews originated, in part, from a subgroup of the Roanoke colonists of 1586. Given this, a new historical narrative of early European colonization in North America during the 1500s is proposed.