Culicoides kingi and Culicoides oxystoma belong to the Schultzei group of biting midges, which are significant vectors of livestock diseases in India. The morphological identification of these species remains challenging due to overlapping characteristics, making species-specific identification crucial for resolving taxonomic ambiguities and understanding their roles in disease transmission. This study aims to develop accurate tools for differentiating C. oxystoma from C. kingi using traditional morphometry and DNA-based identification assays which can be effectively utilized. Specimens were collected from various regions of India. A total of 143 C. oxystoma/C. kingi individuals were identified using a combination of traditional morphometric analyses and PCR targeting the cytochrome c oxidase subunit I gene. Thirteen morphometric measurements were taken from the head, wings, and abdomen of slide-mounted specimens for species discrimination. Five morphometric characteristics such as the antennae, wings, spermathecae, maxillary palp with size and shape of the sensory pit, and the hind tibial comb proved to be effective for species differentiation. Molecular phylogenetic analysis further validated the accuracy of the DNA-based identification approach, demonstrating its efficiency as a simple and rapid molecular identification tool. This study provides the first comprehensive differentiation of both species in India using a combination of morphological and molecular techniques. The application of identification from female genitalia and the DNA-based identification method offers a reliable approach for species identification, which can be instrumental in future epidemiological studies at national and international levels.