COI-based DNA barcoding could be an efficient tool for species identification of deep-sea fishes and could lead to the discovery of cryptic species diversity. However, the availability of reference sequences of deep-sea fishes for DNA barcoding is limited, especially for fishes in the northwestern Pacific Ocean. In this study, we performed DNA barcoding of mesopelagic and demersal fish species on the continental shelf and upper slope, collected from deepwater fisheries around Japan and southern Taiwan, to accumulate the reference sequences of deep-sea fishes in the northwestern Pacific Ocean. Overall, we obtained the COI sequences of 123 species from 50 families. Genetic diversity within each species for which COI sequences were obtained from multiple specimens was examined, and we found that Chimaera phantasma (Chimaeridae), Harpadon microchir (Synodontidae), and Pyramodon ventralis (Carapidae) showed high intraspecific genetic differentiation of more than 2% Kimura two-parameter distance. Moreover, for 19 widespread deep-sea fishes, a comparison between our data and previously acquired COI sequence data suggested a high level (more than 2% Kimura two-parameter distance) of genetic differentiation between the northwestern Pacific Ocean and other oceans in each widespread species. These results suggest that many cryptic species or regional populations have not yet been discovered in deep-sea fishes. Alternatively, genetic differentiation was not found worldwide for six species. These results indicate that many taxonomic and biogeographical issues remain for deep-sea fishes, and our DNA barcoding data would provide better understanding of these issues.