The sea urchin cardinalfish, Siphamia tubifer, inhabits coral reefs throughout the Indo-Pacific and forms a highly specific, bioluminescent symbiosis with Photobacterium mandapamensis, a subspecies of P. leiognathi and a member of the vibrio family. These facultative symbionts are acquired from the environment during larval development and colonize the host’s gut-associated light organ. Despite a high degree of specificity of the S. tubifer-P. mandapamensis symbiosis, there is significant strain-level diversity within an individual host. Using a PCR fingerprinting technique, symbiont strain diversity was characterized and compared between individuals within and between populations of S. tubifer collected from Okinawa, Japan and Verde Island, Philippines. The results indicate that an individual fish associates with a unique set of between two and ten symbiont strains, with little to no overlap in strains between hosts. Additionally, there were no shared strains between fish sampled from Japan and the Philippines. In fact, a whole genome analysis of 72 distinct P. mandapamensis strains revealed genetic differentiation between symbionts from these two locations. Phenotypic assays of the unique strains in culture also indicated key differences, including a trade-off between their luminosity and growth rates. To better understand how these distinct symbiont communities assemble, in vitro competition assays were carried out between strains in a naturally occurring three-member community. Strong priority effects were observed despite differences in their growth efficiencies and competition outcomes. Taken together, these results suggest that strain-level symbiont community composition within a light organ is highly dependent on the timing and location of symbiont acquisition and could have meaningful consequences for the bioluminescent symbiosis with S. tubifer. This study ultimately highlights the importance of investigating sub-species strain-level diversity in microbial symbiosis, even for systems with otherwise seemingly high specificity.