The data presented here is from a study investigating the effect of substituting soybean meal with a newly developed protein source (based on the yeast Cyberlindnera jadinii) in concentrate feed for dairy cows of Norwegian Red breed (NR) on the milk fatty acid composition. A total of 48 NR dairy cows were randomly allocated into three different feeding treatments (16 animals in each group). The feeding treatments consisted of the same basal diet of grass silage, but different concentrate feeds. The experimental concentrate feeds were all based on barley as the main ingredient, but 7 % of the barley (BAR, used as negative control) was replaced by either the conventional soybean meal (SBM, used as control) or the yeast Cyberlindnera jadinii (YEA). The experiment lasted for a total of 10 weeks with the first two weeks considered as an adaptation period where all cows were fed the same control diet (grass silage and SBM), before the cows were allocated to experimental diets for a period of eight weeks. Individual milk samples from weeks 2, 6 and 10 were analyzed for the total fatty acid composition using gas chromatography and mass spectrometry (GC-MS). The amount of each individual fatty acid quantified is given in area (countsmin). The three different feed concentrates (SBM, BAR and YEA) were also analyzed for fatty acid composition, and is given in area (countsmin).