Prochloron is an oxygenic photosynthetic bacterium that lives in obligate symbiosis with didemnid ascidians, such as Diplosoma spp., Lissoclinum spp. and Trididemnum spp. This study investigated the genetic diversity of the genus Prochloron by constructing a phylogenetic tree based on the 16S rRNA gene sequences of 27 isolates from 11 species of didemnid ascidians collected from Japan, Australia and the USA. The 27 isolates formed three phylogenetic groups: 22 of the samples were identified to be closely related members of Prochloron. Two samples, isolated from Trididemnum nubilum and Trididemnum clinides, were found to belong to the species Synechocystis trididemni, the closest relative of Prochloron. Three isolates formed a separate group from both Prochloron sp. and S. trididemni, potentially indicating a new symbiotic phylotype. Genomic polymorphism analysis, employing cyanobacterium-specific highly iterative palindrome 1 repeats, could not delineate the isolates further. For the Prochloron sp. isolates, the phylogenetic outcome was independent of host species and geographic origin of the sample indicating a low level of host specificity, low genetic variation within the taxon and possibly a lack of a host–symbiont relationship during reproductive dispersal. This study contributes significantly to the understanding of Prochloron diversity and phylogeny, and implications for the evolutionary relationship of prochlorophytes, cyanobacteria and chloroplasts are also discussed.