Abstract: Although Blakiston's Fish Owl (Bubo blakistoni) is widely treated as a single species, marked differences in the structure of pair duets between continental and insular populations have been documented. However, no study has quantitatively assessed these vocal differences. We obtained 192 duets from 22 pairs of Blakiston's Fish Owl: 15 pairs of B. b. blakistoni from the Japanese island of Hokkaido and the Russian Kuril island of Kunashir, and seven pairs of B. b. doerriesi from Primorye on the Russian mainland. This is a sizeable dataset for such a large, retiring, and rare owl. We conducted bioacoustic examinations of 14 vocal parameters using principal component analysis and the Isler criterion to quantitatively test species boundaries within the B. blakistoni complex. We found that the insular populations on Hokkaido and Kunashir emerged as vocally similar to each other but markedly different from the continental populations of B. blakistoni, corresponding closely with presently accepted subspecies limits. Bioacoustic differences in the duets of the insular and continental groups are greater than the pairwise comparisons of territorial vocalisations between other sympatric owl species. Based on the reproductive importance of vocal duets in owl biology, we propose the taxonomic elevation of the continental subspecies to species level as Northern Fish Owl B. doerriesi. Our study corroborates the importance of bioacoustics in ascertaining species boundaries in owls and has important implications for the management of the two newly delimited species, each likely to be assessed as Endangered. Both species should be managed independently to optimise conservation outcomes.