Prevalence of infection by bacterial symbionts may reflect their interactions with the host and has been shown to be correlated with environmental factors. Yet, it is still unclear whether infection by symbionts is determined by environmental factors affecting the early or imago stage of the host. Here, we identified and localized the symbiont Candidatus Cardinium hertigii (Bacteroidetes) in sympatric Culicoides biting midge species, examined its abundance, and studied its association with environmental factors. The prevalence of adult infection differed, with 50.7% from C. imicola, 31.4% from C. oxystoma, and 0% from C. schultzei gp., although phylogenetic analyses showed that Cardinium in these species is almost identical. In addition, prevalence of infection differed between climate regions, with lowest prevalence in the arid region and highest prevalence in the Mediterranean region. Multivariate linear regression analysis of Cardinium prevalence together with climatic and satellite imagery data-derived environmental variables revealed that infection prevalence is significantly associated with land surface temperature and explained up to 89.7% of infection prevalence variability. These findings suggest that the observed variation of Cardinium infection of the imago stage of Culicoides may be influenced by environmental conditions during the latter's early developmental stages.