Partial sequences of the cytochrome c oxidase subunlt I (COI) gene were determined for six species of the genus Diplosoma (Ascidiacea, Didemnidae) to develop tools for species identification. Because each Diplosoma species has distinctly different COI haplotype(s), the gene sequence seems to be usable for species discrimination in this ascidian genus. The phylogenetic hypothesis supported by the COI data is congruent with the distribution of character states of the retractor muscle. In this paper, we describe a new Diplosoma species harboring symbiotic cyanophytes, found on Miyakojima Island, Ryukyu Archipelago, Japan. Diplosoma aggregatum sp. nov. forms mosaic-like aggregates of small colonies. Although the zooids of D. aggregatum are similar to those of D. virens, these species are differentiated by colony form and COI sequences.