In the context of emergence or re-emergence of vector-borne diseases, several species of Culicoides Latreille are involved in the transmission of viruses and parasites, affecting animals and humans (respectively) in the Afrotropical Region. Rapid and reliable identification of vector species is required, but morphology based identification requires scarce taxonomic expertise. In addition, systematic and taxonomic classifications are problematic, as authors disagree on placement of certain species into specific subgenera or informal species-groups. Morphological identification of Culicoides larvae is also not possible. This study applied an integrative approach, applying both morphological and molecular criteria to revise the systematics and taxonomy of Culicoides species occurring in the Afrotropics, using a multi-marker molecular phylogeny and species delineation. DNA barcodes were also established for Afrotropical species and the DNA barcoding was tested for species identifications at the larval stage on a large number of samples collected from the Niayes area, Senegal. Results revealed: 1) a new species for science (provisionally named Culicoides sp. # 22) and affiliated to the subgenus Avaritia Fox; 2) the affiliation of the C. similis and C. neavei species-groups to the subgenus Synhelea Kieffer; and 3) the existence of cryptic species within C. oxystoma Kieffer in the subgenus Remmia Glukhova. This study proves the efficiency of DNA barcoding for studying Culicoides larval diversity. This offers opportunities for the monitoring of Culicoides and implementation of in-depth eco-epidemiological studies to better control Culicoides-borne diseases, such as African horse sickness in the Niayes Region, Senegal and in sub-Saharan Africa.