Squids from the genus Euprymna (Cephalopoda: Sepiolidae) and their symbiotic bacteria Vibrio fischeri form a mutualism in which vibrios inhabit a complex light organ within the squid host. A host-mediated daily expulsion event seeds surrounding seawater with symbiotically capable V. fischeri that environmentally colonize newly hatched axenic Euprymna juveniles. Competition experiments using native and non-native Vibrio have shown that this expulsion/re-colonization phenomenon has led to cospeciation in this system in the Pacific Ocean; however, the genetic architecture of these symbiotic populations has not been determined. Using genetic diversity and nested clade analyses we have examined the variation and history of three allopatric Euprymna squid species (E. scolopes of Hawaii, E. hyllebergi of Thailand, and E. tasmanica from Australia) and their respective Vibrio symbionts. Euprymna populations appear to be very genetically distinct from each other, exhibiting little or no migration over large geographical distances. In contrast, Vibrio symbiont populations contain more diverse haplotypes, suggesting both host presence and unidentified factors facilitating long-distance migration structure in Pacific Vibrio populations. Findings from this study highlight the importance of how interactions between symbiotic organisms can unexpectedly shape population structure in phylogeographical studies.