Spatial and temporal genetic structure of the eastern oyster Crassostrea virginica was examined along the coast of North Carolina (NC); utilizing a fragment of the mitochondrial large (16S) ribosomal subunit gene sequence known to distinguish three regional haplotypes in C. virginica—North Atlantic, South Atlantic, and Gulf of Mexico. Significant geographic genetic structure was identified among all populations studied, with little temporal genetic variation observed over the 9-y sampling period. The results are consistent with those of previous studies analyzing geographic variation in mitochondrial DNA16S sequence data, which revealed a genetic discontinuity between North and South Atlantic oyster assemblages along the NC Coast. The increased geographic resolution and duration of sampling in this study revealed a sustained significant shift in genetic composition between Pamlico Sound populations and southern NC populations of C. virginica. These results suggest that a prolonged or persistent barrier impacting larval dispersal or post settlement survival exists between these regions limiting gene flow between North and South Atlantic oyster assemblages.