BackgroundExtensive phenotypic plasticity in oysters makes them difficult to identify based on morphology alone, but their identities can be resolved by applying genetic and genomic technologies. In this study, we collected unknown oyster specimens from Hawaiian waters for genetic identification.MethodsWe sequenced two partial gene fragments, mitochondrial 16S ribosomal RNA (16S) and cytochrome c oxidase subunit I (COI), in 48 samples: 27 unidentified oyster specimens collected from two locations on O‘ahu, 13 known specimens from a hatchery in Hilo, Hawai‘i Island, and 8 known specimens from Hilo Bay, Hawai‘i Island.ResultsMolecular data identified approximately 85% of unknown samples as belonging to the Ostrea stentina/aupouria/equestris species complex, a globally distributed group with a history of uncertain and controversial taxonomic status. The remaining unknown samples were the native Dendostrea sandvichensis (G. B. Sowerby II, 1871), and nonnative Crassostrea gigas (Thunberg, 1793), the latter of which is a commercial species that was introduced to Hawai‘I from multiple sources during the 20th century. Phylogenetic analysis placed Hawai‘i Ostrea alongside samples from China, Japan, and New Zealand, grouping them within the recently classified western Pacific O. equestris. Until now, four extant species of true oyster have been documented in Hawai‘i. This study expands the known range of O. equestris by providing the first verification of its occurrence in Hawai‘i.