The objective of the study was to determine temporal fat deposition and fatty acid profiles in beef cows fed hay- or barley silage-based diets, with or without flaxseed. Crossbred cull beef cows (n = 64, >30 mo of age, 620 ± 5 kg) were removed from grassland pastures, randomly assigned to 16 pens, and given ad libitum access to 50:50 (wt/wt, DM basis) forage:concentrate diets containing 0 or 15% ground flaxseed (DM basis, 5.2% added fat). Diets consisted of hay control (HC), hay+flaxseed (HF), barley silage control (SC), and silage+flaxseed (SF). Backfat biopsies were obtained from each cow at 0, 6, and 12 wk, and at slaughter (~20 wk) to assess fatty acid composition. With the exception of feed efficiency, flaxseed × forage interactions were not significant for backfat accumulation or performance parameters. Flaxseed improved (P < 0.01) feed conversion when supplemented to hay-based diet and increased ADG (P = 0.03), resulting in a heavier (P = 0.02) BW. Compared with hay, barley silage increased (P < 0.01) DMI, ADG, and feed efficiency. Subcutaneous fat contained 0.68% n-3 fatty acids at wk 0, and reached 0.68, 0.81, and 0.94% in HF cows after 6, 12, and 20 wk, respectively (Y(n-3) = 0.0133X + 0.6491, r = 0.87). It was 0.67% at wk 0, and reached 0.65, 0.77, and 0.90% in SF cows after 6, 12, and 20 wk, respectively (Y(n-3) = 0.0121X + 0.6349, r = 0.75). In contrast, weight percentage of n-3 fatty acids decreased in HC cows from 0.63, 0.50, and 0.47, to 0.43%, and in SC cows from 0.63, 0.40, and 0.36, to 0.33% over the 20 wk. A forage × flaxseed interaction (P < 0.05) occurred for many of the α-linolenic acid (ALA) biohydrogenation intermediates, including vaccenic acid (C18:1 trans-11) and CLA (combined C18:2 trans-7,cis-9 and cis-9,trans-11) in plasma, and in subcutaneous fat this also included non-CLA dienes. Concentrations of most α-linolenic acid biohydrogenation intermediates were greater when feeding flaxseed with hay. In conclusion, forage source altered plasma concentrations and rate of accumulation of ALA biohydrogenation products in subcutaneous fat from beef cows fed flaxseed. Factors responsible for this response are yet to be defined, but may include forage-mediated changes in ruminal biohydrogenation of ALA, as well as alterations in fatty acid metabolism and deposition.