Ascidians are one of the most significant individuals of benthic organisms. Their exaggerating recruitment all over the world via their attachments to several natural and artificial substrata has substantially boosted the biodiversity of the marine ecosystem. Morphological identification of ascidian demands highly specialized experience to eliminate them since frequent misidentification. MT-COI gene sequence has been used as a suitable marker for the invertebrate distinctness. In the present study, the Phenol-chloroform method, PCR, and COI sequencing were selected to elucidate the molecular identification of selected ascidians from Egypt and display their phylogenetic relationships with other COIs of ascidian species in the world obtained from GenBank. Constructed phylogenetic trees show that COI could identify the three species: Microcosmus sp. from Ismailia, Symplegma rubra, and Botryllus sp. from Port Said to the closest taxonomic rank.