The objective of the present experiment was to determine if carcass quality and fatty acid profiles of longissimus thoracis (LT) and hamburger would be affected by feeding steers extruded flaxseed on its own followed by hay (non-TMR) compared to when hay and extruded flaxseed were fed together (TMR). Forty-eight steers in six pens were assigned to TMR or non-TMR for an average of 242 days. Dry matter intake was lower for non-TMR versus TMR steers (10.56 vs. 11.42 kg/d; P = 0.02), but final live weight (610 ± 0.50 kg) and average daily gain (1.18 ± 0.02 kg/d) did not differ. Compared to TMR, feeding non-TMR enriched LT and hamburger with α-linolenic acid (ALA; 18:3n-3) by 14%, vaccenic acid (VA; t 11–18:1) by 44%, rumenic acid (RA; c 9, t 11–18:2) by 40%, and conjugated linolenic acid (CLnA) by 58%. Overall, feeding extruded flaxseed separately from hay in a non-TMR was more effective at enhancing deposition of ALA, VA, RA and CLnA in beef. [ABSTRACT FROM AUTHOR]