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The squid-vibrio symbiosis has illuminated fundamental mechanisms of beneficial animal-microbe associations, yet the interactions within sepiolid squid of the Mediterranean Sea remain underexplored. Here we characterize the Sepiola affinis squid-vibrio symbiosis by combining whole-genome sequencing of light-organ isolates, confocal microscopy, and temperature-dependent growth assays. Comparative genomic analyses (ANI, phylogenomics, and functional analyses) revealed two previously undescribed Vibrio species to be symbionts of the S. affinis light organ. One of the species clusters more distantly from other Vibrio species, whereas the second is closer to established Vibrio clades and exhibits an expanded repertoire of mobile elements and Type VI secretion components, suggesting heightened capacity for genetic exchange and interbacterial interaction. Furthermore, confocal microscopy of juvenile squid established that the S. affinis light organ comprises twelve crypts connected by pores and ducts, expanding the number of symbiotic niches relative to other sepiolid squid. In addition, fluorescently labeled isolates from the two Vibrio species colonized juveniles in both mono- and co-colonization patterns within crypts. Finally, growth assays across 16-24°C identified species-specific temperature differences, indicating temperature preferences that may align with seasonal variability in the Mediterranean Sea. Together, these findings position S. affinis to be a tractable model for studying how symbiont diversity, organ architecture, and interbacterial interactions contribute to the stability of a mutualistic symbiosis.