To re-evaluate the taxonomic status of Cheilosia canicularis (Panzer, 1801), C. himantopus (Panzer, 1798) and C. orthotricha Vujić & Claussen, 1994, variation in the mitochondrial DNA (mtDNA) sequence of the cytochrome c oxidase subunit I (COI) gene and 18 nuclear allozyme genes were surveyed in allopatric and sympatric populations from Serbia and Montenegro. Genetic relationships among five populations of these species from the Fruška Gora (Serbia), Kopaonik (Serbia) and Durmitor (Montenegro) mountains were analyzed. Seven allozyme loci (Aat, Aco, Fum, Idh-1, Idh-2, Mdh-2 and Sdh) were diagnostic for delineating C. orthotricha from the other two species, while only a low, but consistent, genetic differentiation was observed between C. canicularis and C. himantopus. Differentiating all three species was possible based solely on the species-specific alleles at the Est-? locus. Sequence comparisons of 738 bp of the COI gene from eleven specimens was consistent with the variability in nuclear allozymes. Sequence data revealed variation in 5% of the nucleotide sites among C. orthotricha and the C. canicularis/C. himantopus pair, while less variation (0.68%) was observed within the pair C. canicularis/C. himantopus. However, the presence of one diagnostic allozyme locus and five consistently variable nucleotide sites in sympatric populations of C. canicularis and C. himantopus (Durmitor, Montenegro) suggest that these two species have separate gene pools.