This article addresses the hypothesis that lactation stage, parity and energy-protein feed additive affect fatty acid composition of blood, liver and adipose tissue of cows. The experiment was conducted on 24 Polish Holstein-Friesian cows divided into two feeding groups. One group of cows was fed solely a total mixed ration, while the other group was fed a ration with the addition of 2 kg of energy-protein supplement per cow/day. During the experiment, the samples of liver, adipose tissue and blood were taken and their fatty acid compositions were determined. Analysis of variance was applied to fatty acid relative weight percentage to determine the effect of the stage of lactation, parity, and energy-protein supplement on the fatty acid composition of the tissues. Stage of lactation had a significant impact on the content of many fatty acids in all examined tissues. We found that parity had no effect on fatty acid composition of blood, whereas it significantly affected C16:1 c9 in liver, and C16:1 c9 and C18:0 in adipose tissue. Energy-protein supplement significantly affected the content of most fatty acids in blood (e.g., C18:1 t11 and C18:3 n-3) and liver (C18:3 n-3, both isomers of conjugated linolenic acid and n-3 fatty acids derived from fish oil), but it did not affect the profile of the adipose tissue of cows. According to our best knowledge, this is the first study showing the relationship between parity, stage of lactation and the composition of fatty acids in blood, liver and adipose tissue of cows.