The two sympatric sibling species Culicoides obsoletus (Meigen) and Culicoides scoticus Downes and Kettle (Diptera: Ceratopogonidae), are known to be competent vectors for bluetongue virus in the Palaearctic region. However, morphological identification of constituent species is only readily applicable to adult males and these two species distinguishing traits have overlapping character states. As their vector competence may differ in space and time, the correct identification and quantification of specimens of both species are essential for understanding bluetongue epidemiology. However, no molecular tools are available for high-throughput identification of the two species. We therefore developed a quantitative duplex real-time PCR assay to determine the relative abundance of each sibling species in a sample using TaqMan probes. For each species, standard curves were constructed from serial dilutions of purified plasmid DNA containing ITS1-5.8S-ITS2 (rDNA) in the range of 10?1 to 10?5 ng/?L. Standard curves were used to quantify samples of mixed C. obsoletus/C. scoticus specimens. Specificity was evaluated with 5156 specimens representing 62 species. Based on the DNA quantities detected according to the standard curves, a quadratic model developed on 1100 males and validated on 555 females was able to predict the relative abundance of each species simultaneously in a one-shot reaction (Pearson coefficient of 0.999). Our assay showed a requirement of two specimens or less for 95% of the predictions, making it highly applicable to field collections. Extensive use of this real-time PCR assay will provide a better understanding of geographical distribution, dynamics, and bionomics on a species level, which is essential for risk assessment. This approach is an important contribution to medical entomology for investigating the vector role of arthropod sibling species.