Beef derived from grass-fed cattle is a specific quality criterion. The effect of grass silage intake on quality characteristics, i.e., fatty acids, fat-soluble vitamins, and lipid-derived volatile composition of intramuscular and perirenal fat from fattening bull weaners were studied. Visible (VIS) and near-infrared (NIR) spectra were also obtained from perirenal fat. Perirenal fat analysis was performed for feeding differentiation purposes. A total of 22 Tudanca breed 11-month-aged bulls were finished on three different diets: grass silage and a commercial concentrate ad libitum (GS-AC), grass silage ad libitum and the commercial concentrate restricted to half of the intake of the GS-AC group (GS-LC), and barley straw and concentrate ad libitum (Str-AC). Feeding had a significant effect (p < 0.05) on γ-linolenic acid and the ratio n-6/n-3 fatty acids. Furthermore, β-carotene content was greater in beef from silage groups than in the Str-AC group. Feeding also affected the perirenal fat composition. Beef from silage-fed bulls and straw-fed bulls could be differentiated by fatty acid percentages, especially 18:0, t-18:1, and c9-18:1, β-carotene content, b* colour value, and carotenoid colour index. However, the VIS or NIR spectra data showed poor differentiating performance, and the volatile composition did not have appreciable differentiation power.