Understanding the population structure of a commercially fished species and how those populations change over time is essential for proper management. Although the blue crab Callinectes sapidus (Rathbun, 1896) covers a large geographic range spanning two continents and several distinct ecosystems, there are no clear morphological characters and a paucity of genetic data that can be used to distinguish populations within and among management areas. In this study, diversity indices were calculated for four loci in the mitochondrial genome of C. sapidus using specimens collected from the Rhode River in Maryland during the summers of 2003 to 2005. The locus with the highest diversity, which occurs within the open reading frame of the nad2 mitochondrial gene, was then used as a marker to determine population structure for samples collected from the Atlantic locations of the Chesapeake Bay; Raleigh, North Carolina; Meadowlands and Tuckerton, New Jersey; and Massachusetts, as well as locations in the Gulf of Mexico spanning the waters of Florida, Alabama, Mississippi, and Texas from 2003 to 2008. For the entire dataset, the genetic variation within each sample site accounted for 99.5%of the total diversity. Also, the Atlantic sampling sites could not be distinguished from the Gulf of Mexico sites using Bayesian inference. Interestingly, the sampling sites from Massachusetts did not show a reduction in their level of genetic diversity despite its location at the northern limit of C. sapidus habitat. There were significant differences looking temporally, however, with the 2003 samples, the year of hurricane Isabel, differing from the 2004 and 2005 samples. Using the nad2 marker along with four microsatellite loci on a sample of 28 megalopae entering the mouth of Chesapeake Bay also demonstrated extremely high diversity at all loci, and the megalopae were significantly different from an adult population of the same year at two of the microsatellite loci. This indicates that genetic diversity is high in adult as well as larval populations and that offshore mixing may be playing a large role in the observed diversity shared among geographically distant habitats. Seasonal weather patterns during larval development followed by selection during the multiple subsequent recruitment events may play a large role in shaping local populations, and larval transport and settling models will provide much more understanding of the population dynamics of this species.