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Background  Biting midge species of the genus  Culicoides  Latreille (Diptera: Ceratopogonidae) comprise more than 1300 species distributed worldwide. Several species of  Culicoides  are vectors of various viruses that can affect animals, like the African horse sickness virus (AHSV), known to be endemic in sub-Saharan Africa. The ecological and veterinary interest of  Culicoides  emphasizes the need for rapid and reliable identification of vector species. However, morphology-based identification has limitations and warrants integration of molecular data. DNA barcoding based on the mitochondrial gene cytochrome  c  oxidase subunit 1 ( cox 1) is used as a rapid and authentic tool for species identification in a wide variety of animal taxa across the globe. In this study, our objectives were as follows: (i) establish a reference DNA barcode for Afrotropical  Culicoides  species; (ii) assess the accuracy of  cox 1 in identifying Afrotropical  Culicoides  species; and (iii) test the applicability of DNA barcoding for species identification on a large number of samples of  Culicoides  larvae from the Niayes area of Senegal, West Africa.    Results  A database of 230  cox 1 sequences belonging to 42 Afrotropical  Culicoides  species was found to be reliable for species-level assignments, which enabled us to identify  cox 1 sequences of  Culicoides  larvae from the Niayes area of Senegal. Of the 933  cox 1 sequences of  Culicoides  larvae analyzed, 906 were correctly identified by their barcode sequences corresponding to eight species of  Culicoides . A total of 1131  cox 1 sequences of adult and larval  Culicoides  were analyzed, and a hierarchical increase in mean divergence was observed according to two taxonomic levels: within species (mean = 1.92%, SE = 0.00), and within genus (mean = 17.82%, SE = 0.00).    Conclusions  Our study proves the efficiency of DNA barcoding for studying  Culicoides  larval diversity in field samples. Such a diagnostic tool offers great opportunities for investigating  Culicoides  immature stages ecology and biology, a prerequisite for the implementation of eco-epidemiological studies to better control AHSV in the Niayes region of Senegal, and more generally in sub-Saharan Africa.    Electronic supplementary material  The online version of this article (10.1186/s13071-018-3176-y) contains supplementary material, which is available to authorized users.

DNA barcoding and molecular identification of field-collected Culicoides larvae in the Niayes area of Senegal