Abstract The fatty acid profile of the longissimus thoracis (LT) muscle and the activity of Δ9 desaturase6, Δ9 desaturase8, and elongase enzymes of beef from feedlot cattle of 2 genetic groups were evaluated. Fifty long-yearling young bulls were divided into two groups following their breed composition: Nellore (n = 25) and Angus × Nellore crossbred (n = 25), were randomly assigned into 4 pens (12 or 13 animals/pen) following a completely randomized design. Animals were sourced from the same breeding season and endured a grazing growing phase in Megathyrsus maximus cv. Mombaça pasture, with protein and energy supplement offered at 0.2% of body weight (BW). The feedlot phase comprehended 105 days of feed, and the diet (80% concentrate) was formulated to meet or safely exceed the beef cattle requirements to target an average daily gain (ADG) of 1.6 kg/day, in which major ingredients were represented by (% DM basis): sorghum silage (20%), ground corn (57.6%), ground soybean seed as the lipid source (20.4%), and a mineral/vitamin premix (2%). Samples of the LT muscle (between the 12th and 13th ribs) were taken right after slaughter to determine the profile of intramuscular fat. Lipids were extracted (2:1 chloroform/methanol), and 5 mL of the lipid extract was used for saponification followed by methylation and submitted for analysis using gas chromatography. The activities of Δ9 desaturase6, Δ9 desaturase8, and elongase enzymes were determined through mathematical indexes, in which their respective saturated and monounsaturated fatty acids were used. Data were analyzed using R (R CORE TEAM, 2021), and the effect of breed on the fatty acid profile was evaluated using the completely randomized design one-way, in which animal was used as the experimental unit. The sum of saturated fatty acids and unsaturated fatty acids was not affected (P ≥ 0.12) by genetic groups. Within FA profiles, crossbred bulls had greater (P = 0.05) concentration of monounsaturated fatty acids (49.7 vs. 52.4%), while beef of Nellore cattle showed greater (P ≤ 0.02) concentration of polyunsaturated fatty acids (6.71 vs. 7.67%) and n-3 (0.53 vs. 0.68%). A tendency (P = 0.07) was observed for the sum of n-6 and n-6/n-3 ratio, in which Nellore cattle had numerically greater values (6.63 vs. 4.89%, and 9.75 vs. 9.22, respectively). The activity of Δ9 desaturase6 and Δ9 desaturase8 was not affected (P ≥ 0.14) by genetic groups, while crossbred animals had greater (P = 0.03) activity of elongase (67.8 vs. 65.6%). Although the overall sum of mono and polyunsaturated fatty acids seems not to be affected, the FA profile and unsaturation/elongation enzymatic activity of beef was influenced by the genetic groups evaluated, in which both Nellore and their F-1 crossbreed counterparts had attractive responses.