Juvenile Atlantic sturgeon (Acipenser oxyrhinchus oxyrhinchus) forming aggregations at coastal sites in the mid-Atlantic Bight were subjected to a mixed stock analysis (MSA) and individual-based assignment using twelve microsatellite loci. We confirmed earlier findings from an analysis of mitochondrial DNA that three river spawning populations (Hudson, James and Delaware Rivers) are the primary sources of these particular marine aggregations. Of the 460 individuals sampled 322, 36 and 47 were assigned to the Hudson, James and Delaware Rivers, respectively. MSA estimated that the New York Bight Distinct Population Segment (Hudson River and Delaware River) contributed 83–90 % of individuals to the marine aggregations and the Chesapeake (James River) and Southeast Distinct Population Segments contributed 5.5–11 %. Mean M-ratios were lower than expected at equilibrium in all three rivers (Delaware = 0.726, Hudson = 0.748, James = 0.664), indicative of genetic bottlenecks affecting all three spawning populations. Further, there were low but detectable levels of inbreeding in all three rivers populations. Effective population size (Ne) was estimated for three populations: Hudson River (172–230 individuals), James River (40–100 individuals) and Delaware River (75–186 individuals). Despite these issues, simulations based on life-history information and the Ne estimates suggest that if ongoing management measures are effective, contemporary levels of population genetic diversity are likely to be retained over the next century.