AbstractCulicoidesbiting midges (Diptera: Ceratopogonidae) are disease vectors responsible for the transmission of several viruses of economic and animal health importance. The recent deployment ofWolbachiawith 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 ofCulicoideshave described the presence ofCandidatusCardinium hertigii (Bacteroidetes). However, the biological impact of this symbiont is yet to be uncovered and awaits a suitable system to studyCardinium-midge interactions. To identify candidate species to investigate these interactions, accurate knowledge of the distribution of the symbiont withinCulicoidespopulations is needed. We used a sensitive nested PCR assay to screenCardiniuminfection in 337 individuals of 25Culicoidesspecies from both Palearctic and Afrotropical regions. Infections were observed in several vector species includingC. imicolaand the pulicaris complex (C. pulicaris, C. bysta, C. newsteadiandC. punctatus) with prevalence ranging from low and intermediate, to fixation. Infection inC. pulicariswas 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 inCulicoides. Through a comparison of our results with previous screens, we evaluate the suitability ofCardinium-infected species for future work pertaining to the symbiont.