A new location record for the night shark, Carcharhinus signatus, is reported for Puerto Rico. DNA barcoding was used to confirm the photo identification of a shark captured incidentally by a small-scale commercial fisher. The adult female night shark was recovered from a depth of 330 m entangled in hook and line gear used in the deep-water snapper and grouper fishery. This record is the first confirmed sighting for Puerto Rico, increasing the shark species reported for the island and supporting its range up to the Mona Passage in the eastern Caribbean Sea.