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Beef knuckles (n = 160) were obtained from source-verified cattle finished on 30% wet distillers grains plus solubles enriched with varying levels of alfalfa hay (4 or 8%), corn silage (6 or 12%), or corn stalks (3 or 6%) based on NDF. Proximate analysis, pH, oxidation-reduction potential, fatty acid composition, and sensory analysis were conducted on the rectus femoris muscle to determine if roughage inclusion, in conjunction with wet distillers grains plus solubles and cattle source, affects beef flavor with particular interest in liver-like off-flavor. Proximate analysis, fat content, and oxidation-reduction potential were unaffected (P ≥ 0.129) by diet or source. For s.c. adipose tissue, cattle from Nebraska (NE) had greater amounts of MUFA (P = 0.048) and unsaturated fatty acids (P = 0.068) but less SFA (P = 0.065) when compared with cattle from South Dakota. Diet affected s.c. adipose tissue levels of 15:0, 17:0, and n-3 fatty acids in which cattle from NE finished on the low corn stalk diet had (P ≤ 0.050) lower levels. Cattle from NE had (P ≤ 0.049) greater i.m. adipose proportions of 13:0 and CLA. Dietary effects (P ≤ 0.050) were observed for i.m. adipose tissue proportions of 16:0, 18:1(n-9), 18:2(n-6), 20:4(n-6), 22:5(n-3), MUFA, PUFA, and n-6 fatty acids. Sensory analysis revealed that cattle from NE were (P ≤ 0.023) less juicy and had less bloody notes when compared with cattle from South Dakota. Cattle finished on the low alfalfa diet were (P ≤ 0.014) more tender and juicy but had more bloody notes. No (P ≥ 0.670) dietary or source effects were noted for liver-like off-flavor. Subcutaneous amounts of 18:2(n-6 trans) (r = −0.17) were inversely related to the incidence of liver-like off-flavor, whereas 20:1(n-9) (r = 0.21), CLA cis-9, trans-11 (r = 0.16) were directly related. Data from this study indicate that type and level of roughage inclusion and cattle source have minimal effects on fatty acid profiles and sensory properties of the musculus rectus femoris. However, individual fatty acids of s.c. and i.m. adipose tissue were significantly correlated with liver-like off-flavor.