The aim of the paper was to extend the knowledge about current fatty acids (FAs) profile of cow milk fat at herds of cows held in dairy farms in Slovakia. 368 milk samples were taken in total from cows of 5 different breeds, namely Holstein (H, n = 105), Red Holstein (R, n = 120), Pinzgau (P, n = 74), Slovak Pied (S, n = 61), and Braunvieh (B, n = 8). Single milk samples were analyzed for fat, protein, lactose content, minerals and fatty acid composition of milk fat using gas chromatography, where 54 FAs were identified and expressed relatively in percentage of peak areas (%) and evaluated in segments in accord with their biosynthetic origine. The combination of acids C12:0, C14:0 and C16:0, as expressed in the sum HCHFA (46.86% for S and 47.35% for P), and also the value of the so-calculated. atherogenic index (2.82 for S and 3.10 for P) are the lowest, and thus the most favorable for human health at the breeds S and P. Medically desirable MUFA (27.11% for the S and 25.84% for P) and PUFA (3.55% for S and 3.26% for P) in these breeds are represented in the highest percentage shares. We conclude that among the observed breeds of dairy cows the Pinzgau and Simental breeds showed a better value of the composition of milk fat from a health perspective.