Cuticular hydrocarbons(CHCs) of the epicuticle wax layer are so far used to differentiate the insects in species and subspecies levels. In this study, four species of malaria vectors(genus Anopheles) were collected from various localities in southern Iran. Twenty specimens of each species were randomly selected and one epicuticular extract was prepared of every five specimens. FID-GC profiles of the extracts did not show any qualitative difference. Using significant difference of CHC mass at retention time(RT) 39.6, the two species of An. sacharovi and An. fluviatilis could be distinguished. Similarly, the two species of An. superpictus & An. sacharovi and An. dthali & An. sacharovi were differentiated by their CHC level at RT 28.5. An. sacharovi was distinguished by integratable peaks at RTs 29.7, 30.6, 30.7, 31 and 32.6 while the other three species just indicated trace peaks at the same RTs. Similarly, An. dthali could be known by an integratable peak at RT 26.2 while An. fluviatilis and An. superpictus indicated trace peaks at the same RT. Integratable peaks and traces at RTs 27.4 and 28.5 were respectively used to differentiate An. superpictus from An. fluviatilis. Lastly, CHC trace amount of An. superpictus at RT 39.6 is another indicator to distinguish it from An. fluviatilis with an integratable peak at the same RT. In harmony with other studies worldwide we hereby report that quantitative analysis of CHCs was successfully applied to differentiate the four Anopheles species of southern Iran.