The biting midge Culicoides imicola Kieffer, 1913 (Diptera: Ceratopogonidae) is a major vector species in the Afrotropical region responsible for Orbiviruses transmission to livestock and equids. Among the most important orbiviroses from a sanitary and economic point of view, Bluetongue (BT) disease affects wild and domestic ruminants, and African Horse Sickness (AHS) s a lethal disease for horses. Historically, C. imicola has an Afrotropical distribution but from 1990s, following epizootic BT outbreaks in the Mediterranean Basin, C. imicola was found on he Mediterranean insular territories and then on the mainland. This leads to the suggestion hat bluetongue emergence in the Mediterranean was due to C. imicola range distribution expansion and global warming. The objective of this study is to investigate the routes and scenarios of colonization of C. imicola in the Mediterranean Basin. We performed a multilocus malysis, using two mitochondrial genes (COI and Cytochrome b), one nuclear gene (Elongation actor alpha) and nine microsatellites markers. Insects were sampled in Africa (12 populations), in the Indian Ocean (3 populations), and in the Mediterranean Basin (30 populations). The routes and scenarios of colonization were tested with an approximate Bayesian computation framework. We found high diversity in Africa and in the east Mediterranean basin compared to the west Mediterranean basin. Haplotype and genotype distribution showed a clear genetic subdivision between the west and east Mediterranean basin. Moreover, the haplotypes and enotypes found in the east Mediterranean basin were genetically related to those found in Africa. The haplotype network and the demographic analyses highlighted a signal of recent population expansion in West Mediterranean zone only. These results support that (1) sub-Saharan Africa is the native range and source of C. imicola for the Mediterranean basin area and (2) there might have been two routes of colonization: one from western