Abstract The aim of the experiment was to define the effect of cattle breed on chemical composition and fatty acid profile in intramuscular fat of Longissimus dorsi muscle of Montbeliarde (M) bulls and Czech Fleckvieh (CF) bulls fattened up to the same live weight and/or age. The pure-bred CF bulls (n = 20) and M bulls (n = 10) were divided into three groups: CF bulls — age (n = 10) fattened up to the age of 572 days; CF bulls — weight (n = 10) fattened up to the live weight of 706 kg and Montbeliarde bulls (n = 10) fattened up to the age of 572 days with live weight of 707 kg. All the bulls were loose-housed in pens with slatted floors from the age of 6 months (after a two-month preparation period) till slaughter and fed an identical feed ration (ad libitum intake of maize silage and a limited amount of concentrate). In both groups of CF bulls the content of C14:0, C14:1, C16:0 and C16:1 was significantly higher and C18:0 and C20:0 content was significantly lower compared to M bulls. However, the overall content of SFA, MUFA and PUFA was not significantly affected by breed. The values of Δ 9-desaturase (16) index were significantly higher in CF bulls than in M bulls. The chemical composition of the Longissimus dorsi muscle was not affected by breed.