Overharvest and habitat alteration have led to a collapse of most commercial Atlantic sturgeon(Acipenser oxyrinchus oxyrinchus)fisheries while pushing the species to rarity or extirpation in most of its historical range. A biologically sound conservation program for this species requires knowledge of its genetic diversity and of the evolutionary relationships among geographic populations. To address these research needs, six microsatellite loci were isolated from A. o. oxyrinchus. Pedigree analysis suggested that all are inherited in a codominant Mendelian pattern. The six loci were tested in ten additional sturgeon species from three genera and three apparent ploidy levels (4n, 8n, 16n). Approximately 70% of successful locus-species amplifications were polymorphic. Polysomy was observed most often in 8n and 16n species. Genetic diversity and population structure of A. o. oxyrinchus were assayed using three polymorphic Aox markers and four markers developed from lake sturgeon (A. fulvescens). A. o. oxyrinchus were sampled from the Altamaha River, Georgia, USA north to the St. Lawrence River, Quebec, Canada. Gulf sturgeon, A. o. desotoi, were sampled from the Suwannee River, Florida, USA, to assess differentiation between the subspecies. Seventy-seven alleles were observed to segregate into unique multilocus genotypes for each of the 392 individuals assayed. Mean diversity was greatest in the Chesapeake Bay (9.7 alleles per locus) and Delaware River (7.4 alleles per locus) collections, and lowest in the St. Lawrence River (4.6 alleles per locus). Mean heterozygosity across seven loci ranged from 44.3% (St. Lawrence River) to 62.6% (Altamaha River). Significant allelic heterogeneity was observed in 82% of pairwise comparisons as well as a global test (p < 0.0001) for A. o. oxyrinchus collections. Genetic distance suggests the presence of at least six subpopulations in A. o. oxyrinchus: St. Lawrence River, St. John River, Hudson River, Delaware River, Albemarle Sound, and Altamaha River. Genetic and geographic distances were positively correlated (r = 0.57, p < 0.03) among A. o. oxyrinchus, suggesting isolation by distance and philopatry. Hierarchical gene diversity analysis indicated significant genetic population structure at every level. Maximum likelihood assignment tests correctly assigned individual fish to collection with a high rate of success (mean = 87.5%); this and other lines of evidence indicated that the Chesapeake Bay collection represents a mixed population of sub-adult sturgeon from northern and southern Atlantic coast populations. Population structure was correlated with that suggested by earlier mitochondrial (mt) DNA analyses. Significant diversity was observed between two Canadian populations from which only a single mt DNA haplotype has been reported.