ABSTRACT: This study was aimed at characterizing four Tunisian pistachio cultivations (Pistacia vera) from the Mateur (North), Nabeul (North-East), Kairouan (Middle) and Sfax (Middle-East) regions and two varieties, Mateur and Ohadi, considering fatty acid composition and main lipid class contents (polar lipids, diacylglycerols, triacylglycerols and free fatty acids) in ripe seeds. Lipid classes were separated using thin layer chromatography, and fatty acid methyl esters (FAMEs) were analyzed by gas chromatography. Oleic acid (C18:19) was the major fatty acid for all samples; those of the Mateur region (the northern region) differed significantly with the lowest content in C18:1 (54.2 % of total fatty acids) and the highest in C18:2 (24.1 %). The total amount of fatty acids (TFA) in the seeds of Ohadi variety was significantly lower than that of the Mateur variety from the Sfax region. Triacylglycerols were most abundant in pistachio seeds from Nabeul (98.5% of total glycerolipids).