We report the results of a survey of the fishes of Kāne‘ohe Bay, O‘ahu, conducted in 2017 as part of the Smithsonian Institution MarineGEO Hawaii bioassessment. We recorded 109 species in 43 families. The most speciose families were Acanthuridae (11 species), Gobiidae (11 species), Pomacentridae (10) and Chaetodontidae (9 species). Nine of the species that we collected are known or suspected to be introduced to the Hawaiian Islands. Specimens were identified, measured and photographed. All specimen vouchers were fixed in formalin and ultimately transferred to 75% ethanol for long-term storage. For nearly all species, we took multiple tissue samples from specimen vouchers prior to formalin-fixation; we preserved tissues in 95% ethanol and then stored them at −80°C. The 5′-end of the mitochondrial cytochrome oxidase I gene (mtCOI) was sequenced for 94 species to confirm their taxonomic identification. Using these barcode sequences, we also measured genetic distances between collected individuals and their conspecifics from other localities outside Hawaii to verify the hypothesis that Hawaiian populations of species broadly distributed throughout the Indo-Pacific may be genetically distinct. We present select case studies to demonstrate the potential for undiscovered endemism in the Hawaiian fish biota.