Genetic variation in eastern oysters (Crassostrea virginica) collected from 13 sites in the Gulf of Mexico was examined using a combination of mitochondrial DNA (mtDNA) sequencing, mtDNA restriction fragment length polymorphism analysis, and nuclear single nucleotide polymorphism analysis. Both mitochondrial and nuclear markers showed significant differentiation among samples. Combined with previous allozyme and microsatellite data, these results indicate considerable population subdivision throughout the Gulf of Mexico, despite the potentially homogenizing effect of larval dispersal.