The determination of the geographical origin of dairy products is an ongoing issue. In this paper the effects of botanical diversity of two pastures on the hydrocarbon and fatty acid composition of cheese fat were studied, over 2 years of experimentation. Two areas in the Italian southwestern Alpine region, dominated by Trifolium alpinum (T) and Festuca nigrescens (F) vegetation, respectively, were chosen, and milk obtained from cows grazing on these pastures was used to produce a semihard traditional cheese. Cheese samples showed a significantly different composition of most linear hydrocarbons, odd-chain (C15, C17, and C17:1) and unsaturated (trans-11,cis-15-C18:2, C18:3, C20:4n-6, C20:4n-3, and 20:5n-3) fatty acids, according to pasture type. The ratio between C(29) and C(27) linear hydrocarbons, unlike the absolute content of the single molecules, showed a good discriminating ability between the two pastures and was little affected by the natural variability due to the climatic and environmental factors.