The eastern oyster, Crassostrea virginica Gmelin, is a common inhabitant of estuarine and coastal waters from maritime Canada through the Gulf of Mexico. Because mitochondrial DNA haplotypes exhibit a distinct genetic break between Atlantic and Gulf oysters at Cape Canaveral, Florida, the degree of divergence between Atlantic and Gulf oysters in nuclear genes is less well known. We examined patterns of variation in four nuclear loci using restriction fragment analysis of amplified DNA (PCR-RFLP) in oysters (n = 317) from 16 locations spanning the geographic range of C. virginica. Marked differentiation was observed between Atlantic and Gulf populations, with smaller differences detected between North Atlantic and South Atlantic populations. Intermediate populations were observed in both eastern and northwest Florida. Regional population structure was also evident in the Gulf Coast, with Texas oysters highly divergent from all other populations.