From the gene collection of the Viticulture Research Station Karlštejn samples of seeds of selected grapevine varieties were obtained during the harvest of 2011 and 2012. Average oil content in analysed grapevine varieties in 2011 was 11.60±0.33 g/100g seed dry matter. Linoleic acid was the most abundant fatty acid in all analysed grape seed oils, contributing between 68.10 g/100 g oil and 78.18 g/100g oil. Linolenic acid was present only in small trace quantities ranging from 0.29 g/100g to 0.77 g/100g oil. Oleic acid content conformed to MUFA content, which ranged from 8.82g/100g to 16.92g/100g. SFA ranged between 9.04g/100g and 12.82g/100g of TFA. Statistical analysis revealed close correlation between PUFA and linoleic acid (R2=0.998) and MUFA and oleic acid content (R2=0.994). Variety of cultivation showed significant impact on the content of fatty acids in oil. Principal component analysis revealed differences or similarity of analysed grapevine varieties related to the content of major FA. The year of cultivation showed different effect on individual FA content.