The presence and diversity of free-living dinoflagellates belonging to the endosymbiotic genus Symbiodinium were explored in seawater samples collected above coral reefs in Kane'ohe Bay, O'ahu, Hawai'i, and Puerto Morelos, Quintana Roo, Mexico. Two genetic markers were used to assess Symbiodinium diversity in the water column: the internal transcribed spacer 2 region of the nuclear ribosomal array (ITS2), and a hypervariable region in domain V of the large subunit (23S) of the chloroplast ribosomal array (cp23S-HVR). Sequencing of cloned gene fragments reveals that clades B, C, D, and H Symbiodinium are detectable in the seawater samples. In addition to the previously described types B1, C3, C15, C21, and D1, novel Symbiodinium sequences belonging to clades B and C were also retrieved. The majority of Symbiodinium sequences recovered from Kane'ohe Bay belonged to clade C and those from Puerto Morelos to clade B, a pattern that reflects the dominant types of Symbiodinium found in endosymbiosis with scleractinian corals in these two areas. This study represents the first direct assessment of Symbiodinium diversity in waters adjacent to coral reefs located in the Caribbean and the Pacific and confirms the presence of Symbiodinium in this compartment of the ecosystem. These data provide context for future studies examining spatial and temporal patterns in the availability of Symbiodinium in the water column, work that will ultimately promote a greater understanding of the interactions between symbiotic dinoflagellates and their environmentally sensitive benthic hosts.