The dinoflagellate Symbiodinium is well known to engage symbiosis with various marine animals, including corals. Recent records of environmental Symbiodinium (occurring in the environment and separately from host animals; usually referred to as ‘free-living’ Symbiodinium) are of special interest, since these environmental populations are essential as symbiont sources for many host animals. In the present study, we carried out a phylogenetic analysis of environmental Symbiodinium isolates (culture strains) from sand, tide pools, or macroalgal surfaces, and environmental DNA clones extracted from the water-column, at numerous sites around Japan. Our phylogenetic analysis based on the nuclear rRNA gene (internal transcribed spacers -1, -2, and 5.8S), indicated that most of the environmental isolates form monophyletic subclades within the Clade-A lineage, and separate from a host-associated Clade-A population with high bootstrap values. Results of the partial nuclear 28S rDNA phylogeny and thecal-plate observations revealed that these environmental isolates were closely related to a previously-described ‘planktonic species’, Symbiodinium natans Gert Hansen et Daugbjerg, which was isolated from a plankton-net sample from the Northeast-Atlantic Ocean. On the other hand, the environmental DNA clones were also noted to be mostly nested within host-associated Symbiodinium groups scattered in various clades. These results led to the assumption that the environmental Symbiodinium can be divided into two groups. One group, as typified by environmental isolates in the present study and previous reports, may be exclusively free-living; the other group exists transiently in free-living forms, possibly having been expelled from animal hosts. The populations within the latter group probably represent environmental sources of viable symbionts, because these are normally host-associated. However, the Symbiodinium in the former group are not expected to engage in stable symbioses with host cnidarians.