The two Sotalia species (the marine S. guianensis and the freshwater S. fluviatilis) have only recently been recognized, and both face several conservation challenges. We investigated the existence of hybridization between the two species in their possible area of sympatry in the Amazon Estuary, in northern Brazil. A fast and cheap PCR-RFLP diagnostic method using nuclear DNA was developed to discriminate between the two species, while allowing the detection of hybrids. All samples that could be identified (N = 51) were identified as S. guianensis, and no hybrids were detected. Our results, coupled with previous mitochondrial data, suggest that S. fluviatilis is not present in the Amazon delta. Thus, sympatry with S. guianensis, if it does occur, may be restricted to upstream areas of the Amazon River.