Abstract Bluetongue is a non‐contagious viral disease causing significant economic losses throughout the world. The bluetongue vectors Culicoides oxystoma and Culicoides actoni, which play a significant role in the transmission of various pathogens, are distributed across different geographical realms. Adults are minute in size with wide phenotypic variation, so morphology‐based species identification is severely constrained by preparatory time and shortage of taxonomic expertise. To make the identification process rapid and effective, a specific primer was designed for the identification of C. actoni based on the multiple sequence alignment of ITS1 sequences of 11 Culicoides species. Along with this, a refined version of existing C. oxystoma specific primer was proposed. The primer sets distinguished C. oxystoma and C. actoni from a pooled sample consisting of other Culicoides species as well as closely related genera such as Forcipomyia and Alluaudomyia. Our findings suggest that the primers were species specific, sensitive and have potential to discriminate vector species C. oxystoma and C. actoni from pooled samples. To the best of our knowledge, these are the first ITS1 sequences generated and submitted in GenBank for Culicoides innoxius, Culicoides shortti, Culicoides palpifer and Culicoides anophelis and the first for Culicoides peregrinus, Culicoides fulvus and C. actoni from India.
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