Many morphologically similar species of the simuliid (Diptera: Simuliidae) subgenus Wilhelmia, Enderlein are difficult to distinguish. Thus, the revision of the subgenus using various morphological, cytogenetic, and genetic analyses has been attempted. Neglected until now, the Balkan Peninsula, a crossroad between Europe and Anatolia, provides insight which could resolve problematic interrelationships of the taxa within this subgenus. To uncover the status and relations within the subgenus Wilhelmia, mtDNA was extracted from 47 individuals of six morphospecies: Simulium balcanicum (Enderlein, 1924), Simulium turgaicum Rubtsov, 1940, Simulium lineatum (Meigen, 1804), Simulium pseudequinum Séguy, 1921, Simulium equinum (Linnaeus, 1758), and Simulium paraequinum Puri, 1933 from 21 sites throughout the Balkan Peninsula. Phylogenetic analysis of the Wilhelmia species using mitochondrial DNA barcoding (COI) gene showed two major branches, the lineatum branch, which includes the lineages sergenti, paraequinum, and lineatum, and the equinum branch. In the equinum branch, the mtDNA sequences formed six clades, with high genetic distances, suggesting the existence of different species. Historically, the clades of the equinum branch appeared at numerous islands, perhaps as a result of allopatric speciation. The paraequinum lineage (lineatum branch) is composed of two species. However, six clades of the lineatum lineage overlapped with intra- and interspecific genetic distances. Our results revealed that the species S. balcanicum, S. pseudequinum B, and S. equinum were omnipresent in the Balkans. The results point to not only the fair diversity of Wilhelmia species in the Balkans, but also indicate that most Wilhelmia species live in sympatry.