Identification of Culicoides (Diptera: Ceratopogonidae) biting midges to species has become important due to their potential role in the transmission of arboviruses such as bluetongue virus, bovine ephemeral fever virus, Akabane virus, African horse sickness virus, epizootic haemorrhagic disease virus and Schmallenberg virus. In several studies, molecular tools, used for the identification of biting midges, revealed the presence of cryptic and undescribed species especially within Pulicaris complex. The presence of cryptic species within species complexes raise questions about their role in viral disease transmission as there are apparent differences in the vectorial capacity between closely related species. In this study, we analyzed the mitochondrial DNA cytochrome oxidase I (COI) gene sequences of species within the Pulicaris complex present in Turkey and determined their phylogenetic relationships. Twenty-one haplotypes within the already described species C. pulicaris P1, C. lupicaris, C. lupicaris L2, C. newsteadi, C. newsteadi N1, C. punctatus, C. fagineus F2 and C. flavipulicaris were determined from the study areas. The molecular analysis revealed further two haplotypes belonging to new non-described cryptic species named as C. lupicaris L3 and Culicoides WBS corresponding to C. lupicaris and Fagineus complex which diverged by 17.9% to 25.7% and 18.7% to 31.8%, respectively from other species in the subgenus Culicoides. Genetic divergence within species was <2.0% and phylogenetic analyses of the COI dataset revealed 22 different monophyletic separate clades within two major cluster. The results of this study emphasize the applicability of COI sequences as a diagnostic marker for differentiating Culicoides species and revealing cryptic species.