Most deep-water chondrichthyans occurring off mainland Portugal are distributed worldwide. There are many closely related species with similar morphology, and the lack of diagnostic characters that unambiguously allow species discrimination has led to extensive identification problems. Here, DNA barcodes were used to evaluate the suitability of cytochrome c oxidase I (COI) to identify deep-water chondrichthyans and to further contribute to the worldwide molecular databases currently in use. The sample was composed of 40 individuals from the families Chimaeridae, Centrophoridae, Somniosidae, Etmopteridae, Oxynotidae, Dalatiidae, Scyliorhinidae, Mitsukurinidae, Chlamydoselachidae and Hexanchidae. COI proved to be adequate for species identification, with almost all putative species recovered as well supported monophyletic clades, with low intraspecific variation. Sequence matches against the BOLD database provided evidence for the existence of identification problems particularly within the genera Hydrolagus, Galeus and Centrophorus. Present results suggest that three different Centrophorus species occur off the Portuguese mainland, C. squamosus, C. granulosus and C. uyato. The sample also included an albino Deania calcea and a C. squamosus specimen without dermal denticles. This study updates the current knowledge on diversity and distribution of deep-water chondrichthyans occurring along the Portuguese coast, and provides an essential tool for species identification and consistency of taxonomic designations worldwide.