Hundreds of ribosomal transcribed spacer sequences (ITS1 and ITS2) distinguish a large diversity of Symbiodinium with distinct ecological and biogeographical distributions, yet formal species assignments remain a subject of controversy. Here we employed a multi-locus approach to test the hypothesis that two Symbiodinium with distinct depth distributions, yet similar ITS sequences, are separate species. In this study, we have employed a novel method of marker development to create microsatellite loci for a host and its symbiont simultaneously. Using these newly developed nuclear microsatellite loci and sequence differences in the psbA chloroplast gene were evaluated under the null hypothesis of panmictic mating within species. We found that geographically separate populations of Symbiodinium of the same depth distribution were more similar genetically to each other than sympatric populations analyzed across depth. While the host showed no population structure across depth of geography. Suggesting that the one host may associate with multiple, closely related symbionts across a depth distribution, and these symbionts might be specifically adapted to a depth zone. By extrapolating from this example, there are 100â€™s of morphologically cryptic yet genetically divergent Symbiodinium spp. awaiting description. These data further substantiate a growing awareness that these microbial eukaryotes are considerably more diverse than current assessments indicate. The modernization of protocols for validating new species is long overdue.