Stranded cetaceans (whales, dolphins, and porpoises) are frequently used to obtain data on species occurrence and demographic trends. Accurate species-level identification of these individuals is crucial, but often challenging or impossible when relying solely on morphological features (e.g., for highly decayed specimens). To aid in the development of a reliable molecular assay for cetacean DNA-based identification, we tested the efficacy of the standardized DNA barcode segment of the coxI gene in identifying cetaceans occurring off the Brazilian coast and in its continental waters. We generated coxI sequences from 150 specimens (collected by 16 Brazilian institutions), most of which included voucher material (skulls, skeletons and/or images) deposited in scientific collections. This allowed a direct comparison between their morphological and molecular identification. CoxI sequences correctly identified ~93% of the samples, comprising 33 species (70% of the 47 cetaceans reported for Brazilian waters). Two species (Berardius arnuxii and Phocoena dioptrica) were sequenced for coxI for the first time. For only two dolphins (Stenella coeruleoalba and S. clymene) and a right whale (Eubalaena australis), coxI failed to identify the species due to overlapping distributions of intra- vs. interspecific divergences. Only one right whale species occurs in the southern hemisphere, facilitating identification in this case. Stenella dolphins present extensive sympatry and potential inter-species hybridization, suggesting that nuclear markers may be required for their reliable identification. These results indicate that DNA barcoding can reliably identify most stranded cetaceans and highlight the importance of voucher materials to validate the construction of a reliable DNA-based identification system.