Holothurians are one of the most dominant taxa in marine communities in the Red Sea and Gulf of Aqaba. However, despite their wide distribution their taxonomic diversity is poorly understood. Recent studies have shown that many species thought to have broad distributions are actually a complex of allied species, with long standing questions regarding the identification of certain sea cucumber species. A total of 140 specimens of sea cucumber tissues were collected for DNA examination. A total of 18 sea cucumber species were identified along the Egyptian coast of the Red Sea and Gulf of Aqaba; Holothuria spinefra; Holothuria sp.; Holothuria leucospilota; Holothuria impatiens; Holothuria nobilis; Holothuria fuscogilva; Holothuria scabra; Holothuria atra; Actinopyga mauritiana; Actinopyga sp. Nov; Actinopyga crassa; pearthonthuria graeefei; Bohadchia tenussima; Bohadchia marmorata; Bohadchia cousteaui; stichopus sp. and Synaptula sp. These species were distinguished using morphological techniques. The DNA sequences of COI gene produced 17 different distinctive clades of sea cucumber. The use of DNA barcoding of the mitochondrial COI gene proves to be a good technique in resolving long standing problems in the identification of sea cucumber.