Abstract. The aim of the study was to determine the effect of cow breed, season and type of diet on the fatty acid (FA) profile of raw milk. A 2-year study was conducted on bulk milk samples collected from eight herds consisting of Czech Fleckvieh (CF, four herds) and Holstein (H, four herds) breeds. One half of the herds of each breed was grazed (G), while the other half was not (N). Samples were collected twice in winter (W) and twice in summer (S). Milk yield in CF (5385.50 kg) was lower than in H (7015.15 kg, P <  0.05). The effect of breed was found in odd-chain, branch-chain and hypercholesterolemic FAs (P <  0.05). The content of fat was lower in summer (S) than in winter (W), being 3.71 and 3.91 g 100 g⁻¹, respectively (P <  0.05). The proportion of saturated and polyunsaturated FAs was lower in S than in W (P <  0.05). The content of monounsaturated FAs was higher in S (30.69 g 100 g⁻¹) than in W (27.72 g 100 g⁻¹, P <  0.05). Milk yield in grazing herds (G, 5197.50 kg) was lower (P <  0.05) than in non-grazing herds (N, 7203.75 kg). The sum of saturated and hypercholesterolemic FAs was lower and the sum of monounsaturated and odd-chain FAs was higher in G than in N (P <  0.05). Content of conjugated linoleic acid (CLA) and C18:3n3 was higher in G (0.93 and 0.64 g 100 g⁻¹) than in N (0.42 and 0.39 g 100 g⁻¹, respectively, P <  0.001).