The spotted seatrout, Cynoscion nebulosus (Cuvier, 1830), lives almost entirely within natal estuaries, and although many genetic studies identify fine-scale population structure, they do not provide a consensus as to the number of stocks, the stock boundaries, or their connectivity. Two perceived limitations of previous studies were addressed. To address a presumption of limited sampling, we assembled a large sample size with broad geographic range: 547 specimens from 18 sampling areas (mean inter-area distance = 270 km) between Texas and North Carolina, representing the majority of this species' range. To address the presumptive limitations of genetic markers, two genetic marker types were compared: a 335-base-pair segment of the mitochondrial DNA (mtDNA) control region and 38 microsatellite loci. MtDNA haplotype frequencies were different only between populations of the Gulf of Mexico (GoM) and the Atlantic Ocean, but not between the western GoM and the eastern GoM. In contrast, the microsatellite loci characterized the species' range-wide population structure as three geographically non-overlapping clusters. These clusters were defined by two genetic breaks: one at the Apalachicola River, on Florida's Gulf Coast, and another in the Atlantic Ocean between Miami and Palm Beach on Florida's east coast. Moreover, within each genetic cluster (identified by microsatellite markers), a pattern of isolation by distance was evident. This new characterization of spotted seatrout population structure supports assessment and management of the species by individual states and defines distinct stock boundaries in Florida, the only state with multiple genetic stocks, as defined by either genetic marker type.