The phylogenetic relationships between naturally occurring Atlantic Crassostrea oyster species were inferred through analyses of mitochondrial (cytochrome oxidase subunit I and 16S) and nuclear (second internal transcribed spacer) sequences. We also scored 15 allozyme loci on 422 oysters to study population structuring of C. rhizophorae and C. brasiliana along 9000 km of the Western Atlantic coastline. Despite morphological similarities, C. virginica was genetically more closely related to C. rhizophorae than to C. brasiliana. In contrast, C. paraibanensis was genetically indistinguishable from C. brasiliana, which is probably a junior synonym of the African C. gasar. Significant genetic differentiation between populations of C. rhizophorae and C. gasar were found along the Western Atlantic coast, supporting an isolation-by-distance pattern.