A protocol that takes advantage of length heteroplasmy in domain V of chloroplast large subunit (cp23S)-ribosomal DNA to identify members of the symbiotic dinoflagellate genus Symbiodinium is presented. This protocol is highly specific for Symbiodinium, can provide intercladal and intracladal identification of a particular Symbiodinium isolate, and can detect multiple Symbiodinium chloroplast genotypes simultaneously in the same isolate, making his technique attractive for a variety of research questions. We used this technique to characterize variation among Symbiodinium populations associated with a range of phylogenetically diverse and geographically discrete hosts. We also examined symbiont variation within a single host, the Caribbean gorgonian Pseudopterogorgia elisabethae, from 9 sites in the Bahamas, and we report a previously undocumented level of symbiont specificity for particular members of Symbiodinium clade B in this gorgonian.