At one time, Atlantic sturgeon supported a signature fishery in the Hudson River Estuary and identification of its migratory patterns is listed as a priority under Long Range Target 1 of the Actions Planned for 2010-2014 (Effectively Managing Migratory Fish). This study provided important new information that will be used by the NYSDEC and NOAA’s Office of Protected Resources to manage Atlantic sturgeon in the Hudson River ecosystem and coastwide. Atlantic sturgeon is federally listed under the U.S. Endangered Species Act (ESA) as five Distinct Population Segments (DPS), of which four were designated as “endangered” and one as “threatened.” The New York Bight DPS is comprised of the Hudson and Delaware River populations and is listed as “endangered.” Subadult Atlantic sturgeon are known to exit their natal estuaries to coastal waters and non-natal estuaries where they are vulnerable to distant anthropogenic threats. In fact, during the warmer months, the Hudson River hosts large numbers of subadults, but their population and DPS origin is largely unknown although Section 7 of the ESA demands that origin of individual specimens be determined. We used microsatellite DNA analysis at 11 loci and sequence analysis of the mitochondrial DNA (mtDNA) control region to determine the DPS and population origin of 106 subadult Atlantic sturgeon collected in the lower tidal Hudson River estuary. We found that 101 of the 106 subadults assigned to the Hudson River with at least 95% and usually 100% probability. Of those 5 specimens that did not assign to the Hudson, 2 assigned to the James River, VA, 2 assigned to the Kennebec River, ME, and 1 assigned to the Saint John River, NB. Thus, four specimens assigned to DPS other than the New York Bight DPS and one to the Canadian Management Unit. This analysis will permit the quantification of the effects of anthropogenic threats in different locales or across seasons in the Hudson River Estuary on individual populations or DPS of Atlantic sturgeon and will serve as a model for similar population composition analysis for other estuaries coastwide.