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Genetic equilibrium of polymorphic enzyme loci from four natural populations of Cheilosia vernalis was tested. The study populations were from different regions of the Balkan Peninsula: Mediterranean area (Morinj, Montenegro: CVMOR), low mountain in the Pannonian plain (Fruska Gora, Serbia: CVFG), and two high Dinaric mountains (Durmitor, Montenegro: CVDUR; and Kopaonik, Serbia: CVKOP). Out of twelve enzyme loci analyzed for genetic variability, only four to six were polymorphic in the studied populations. For those loci, the observed and expected values of genotype frequencies were compared with chi-square goodness-of-fit tests. Analysis of deviations of phenotypic classes from Hardy-Weinberg equilibrium revealed significant differences at polymorphic loci of all populations except in CVKOP. This implied possible important influence of evolutionary mechanisms such as low migration rates, population substructuring and natural selection in creation and maintenance of genetic variability.