Atlantic sturgeon Acipenser oxyrinchus is large, long-lived, and anadromous with subspecies distributed along the Atlantic (A. oxyrinchus oxyrinchus) and Gulf of Mexico (A. o. desotoi) coasts of North America. Although it is not certain if extirpation of some population units has occurred, because of anthropogenic influences abundances of all populations are low compared with historical levels. Informed management of A. oxyrinchus demands a detailed knowledge of its population structure, levels of genetic diversity, and likelihood to home to natal rivers. We compared the use of mitochondrial DNA (mtDNA) control region sequence and microsatellite nuclear DNA (nDNA) analyses in identifying the stock structure and homing fidelity of Atlantic and Gulf coast populations of A. oxyrinchus. The approaches were concordant in that they revealed moderate to high levels of genetic diversity and suggested that populations of Atlantic sturgeon are highly structured. At least six genetically distinct management units were detected using the two approaches among the rivers surveyed. Mitochondrial DNA sequences revealed a significant cline in haplotype diversity along the Atlantic coast with monomorphism observed in Canadian populations. High levels of nDNA diversity were also observed among populations along the Atlantic coast, including the two Canadian populations, probably resulting from the more rapid rate of mutational and evolutionary change at microsatellite loci. Estimates of gene flow among populations were similar between both approaches with the exception that because of mtDNA monomorphism in Canadian populations, gene flow estimates between them were unobtainable. Analyses of both genomes provided high resolution and confidence in characterizing the population structure of Atlantic sturgeon. Microsatellite analysis was particularly informative in delineating population structure in rivers that were recently glaciated and may prove diagnostic in rivers that are geographically proximal along the south Atlantic coast of the US.