A morphology-based barcoding library of market teleost fishes (Teleostei) in Cebu is built based on cytochrome c oxidase subunit I (COI) sequences and voucher specimens which aimed to establish a reliable reference of frequently traded fishes in the province, a biodiversity hotspot at the center of the Philippine archipelago. A total of 1721 specimens were collected from 18 fish markets and landing sites around the province, in which 538 specimens were sequenced belonging to 393 species from 229 genera, 86 families, and 37 orders. Most speciose families are coral reef or reef-related shallow-water species. Twelve species from 11 families are newly recorded in the Philippine waters, among which 7 species are deep-sea inhabitants, while 3 species have expanded their distribution range. Only 20 taxa could not be identified to the species level due to the difficulty in morphological examinations, absence of matched reference sequences in online databases, and/or problematic species awaiting further studies. This first comprehensive DNA barcoding survey of Cebu fishes can facilitate further taxonomic research as well as the conservation and management of fisheries in the Philippines.