Acaryochloris spp. are unique cyanobacteria which contain chlorophyll d as the predominant pigment. The phylogenetic diversity of Acaryochloris spp. associated with 7 Prochloron- or Synechocystis-containing didemnid ascidians and 1 Synechococcus-containing sponge obtained from the coast of the Republic of Palau was analyzed; we established a PCR primer set designed to selectively amplify the partial 16S rRNA gene of Acaryochloris spp. even in DNA samples containing a large amount of other cyanobacterial and algal DNAs. Polymerase chain reaction-denaturing gradient gel electrophoresis with this primer set enabled detection of the phyogenetic diversity of Acaryochloris spp. All the ascidian and sponge samples contained Acaryochloris spp. Fourteen phylotypes that were highly homologous (98–100%) with A. marina MBIC11017 were detected, while only 2 phylotypes were detected with our previously developed method for detecting cyanobacteria. The results also revealed that many uncultured phylotypes of Acaryochloris spp. were associated with those didemnid ascidians, since a clonal culture of only 1 phylotype has been established thus far. No specific relationship was found among the Acaryochloris phylotypes and the genera of the ascidians even when sample localities were identical; therefore, these invertebrates may provide a favorable habitat for Acaryochloris spp. rather than hosts showing any specific symbiotic relationships.