Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are insect vectors of economically importantveterinary diseases such as African horse sickness, bluetongue, and Schmallenberg virus. The identification of Culicoides basedon morphological features can be difficult.Three species of biting midges, Culicoides nubeculosus, C. stigma, and C. parroti haveemerged in the laboratory from mud collected around watering troughs on a farm in northern France. Emerging Culicoides werecharacterized morphologically and molecularly using molecular markers. The closely related species C. stigma and C. parrotishowed highly divergent sequences for both mitochondrial (cytochrome B and cytochrome oxidase I) and ribosomal DNA firstinternal transcribed spacer. A RFLP based on a single restriction using the same enzyme (HaeIII) for both cytochrome C oxidaseI and cytochrome B is proposed to identify these species.