Culicoides imicola is the main vector for bluetongue (BT) and African horse sickness (AHS) viruses in the Mediterranean basin and in southern Europe. In this study, we analysed partial mitochondrial cytochrome c oxidase subunit I (COI) gene to characterize and confirm population expansion of Culicoides imicola across Spain. The data were analysed at two hierarchical levels to test the relationship between C. imicola haplotypes in Spain (n=215 from 58 different locations) and worldwide (n=277). We found nineteen different haplotypes within the Spanish population, including 11 new haplotypes. No matrilineal subdivision was found within the Spanish population, while western and eastern Mediterranean C. imicola populations were very structured. These findings were further supported by median networks and mismatch haplotype distributions. Median networks demonstrated that the haplotypes we observed in the western Mediterranean region were closely related with one another, creating a clear star-like phylogeny separated only by a single mutation from eastern haplotypes. The two, genetically distinct, sources of C. imicola in the Mediterranean basin, thus, were confirmed. This type of star-like population structure centred around the most frequent haplotype is best explained by rapid expansion. Furthermore, the proposed northern expansion was also supported by the statistically negative Tajima's D and Fu's Fs values, as well as predicted mismatch distributions of sudden and spatially expanding populations. Our results thus indicated that C. imicola population expansion was a rapid and recent phenomenon.