AbstractCulicoides biting midges (Diptera: Ceratopogonidae) are disease vectors responsible for the transmission of several viruses of economic and animal health importance. The recent deployment of Wolbachia with pathogen-blocking capacity to control viral disease transmission by mosquitoes has led to a focus on the potential use of endosymbionts to control arboviruses transmitted by other vector species. Previous screens of Culicoides have described the presence of Candidatus Cardinium hertigii (Bacteroidetes). However, the biological impact of this symbiont is yet to be uncovered and awaits a suitable system to study Cardinium-midge interactions. To identify candidate species to investigate these interactions, accurate knowledge of the distribution of the symbiont within Culicoides populations is needed. We used a sensitive nested PCR assay to screen Cardinium infection in 337 individuals of 25 Culicoides species from both Palearctic and Afrotropical regions. Infections were observed in several vector species including C. imicola and the pulicaris complex (C. pulicaris, C. bysta, C. newsteadi and C. punctatus) with prevalence ranging from low and intermediate, to fixation. Infection in C. pulicaris was very rare in comparison to a previous study, and there is evidence the prior record of high prevalence represents a laboratory contamination error. Phylogenetic analysis based on the Gyrase B gene sequence grouped all new isolates within “group C” of the genus, a clade which has to date been exclusively described in Culicoides. Through a comparison of our results with previous screens, we evaluate the suitability of Cardinium-infected species for future work pertaining to the symbiont.