Atlantic Sturgeon is listed under the U.S. Endangered Species Act as five Distinct Population Segments (DPS). The "endangered" New York Bight (NYB) DPS is thought to only harbor two populations; one in the Hudson River and a second smaller one in the Delaware River. Historically, the Connecticut River probably supported a spawning population of Atlantic Sturgeon that was believed extirpated many decades ago. In 2014, we successfully collected pre-migratory juvenile specimens from the lower Connecticut River which were subjected to mitochondrial DNA (mtDNA) control region sequence and microsatellite analyses to determine their genetic relatedness to other populations coastwide. Haplotype and allelic frequencies differed significantly between the Connecticut River collection and all other populations coastwide. Sibship analyses of the microsatellite data indicated that the Connecticut River collection was comprised of a small number of families that were likely the offspring of a limited number of breeders. This was supported by analysis of effective population size (Ne) and number of breeders (Nb). STRUCTURE analysis suggested that there were 11 genetic clusters among the coastwide collections and that from the Connecticut River was distinct from those in all other rivers. This was supported by UPGMA analyses of the microsatellite data. In AMOVA analyses, among region variation was maximized, and among population within regions variation minimized when the Connecticut River collection was separate from the other two populations in the NYB DPS indicating the dissimilarity between the Connecticut River collection and the other two populations in the NYB DPS. Use of mixed stock analysis indicated that the Connecticut River juvenile collection was comprised of specimens primarily of South Atlantic and Chesapeake Bay DPS origins. The most parsimonious explanation for these results is that the Connecticut River hosted successful natural reproduction in 2013 and that its offspring were descendants of a small number of colonizers from populations south of the NYB DPS, most notably the South Atlantic DPS. Our results run contrary to the belief that re-colonizers of extirpated populations primarily originate in proximal populations.