The fatty acid profile of Greek buffalo milk samples was significantly influenced by the lactation stage and/or environmental temperature. Thus, during the 6th, 7th and 8th month after parturition (colder winter months), the fatty acids with lower melting point (unsaturated) were detected at higher concentrations in order to contribute in the reduction of melting point of total lipid. This phenomenon probably occurred via enzymes which function in elongation and desaturation of fatty acids. The important fatty acids butyric (C4:0), vaccenic (C18:1ω-7 trans-11) and rumenic (C18:2 cis-9, trans-11) remained stable throughout the study. Cholesterol content was detected at lower levels in the milk of buffalo samples than in other kinds of milk.