This study investigated the existing species of Crassostrea in the natural environment, farming systems and artificial spat collectors at a protected estuarine area in Brazil, through PCR-RFLP of mitochondrial 16S rRNA. Among 450 samples collected in the natural environment, 303 were C. brasiliana and 147 C. rhizophorae. Oysters present in the rocky subtidal zone were C. brasiliana. However, both species occurred on mangrove roots in the intertidal zone. Farm-raised samples included only C. brasiliana. It was observed that attached specimens in commercial collectors had a banding pattern distinct from C. brasiliana and C. rhizophorae, indicating the presence of a third species in the estuary. The 16S rRNA sequence analysis showed that these specimens are clustered with the oysters from Pacific and Indian Oceans, and genetically close to the oysters of Beihai, China (0.3% genetic distance). Oysters obtained from the seed capture showed 17.8% distance of in relation to C. brasiliana, 17.6% for C. rhizophorae and 10.3% for C. gigas, demonstrating high genetic divergence from these species. The occurrence of an exotic species in the Cananéia estuary may have strong ecological and economic implications which require new guidelines for farming, conservation and sustainable fisheries management for the native oyster species.