Mitochondria are cell organelles vulnerable to stress and may be damaged either by reactive oxygen or nitrogen species during the stress response. The antioxidant tocopherol may be useful in protecting mitochondrial health. The objective of our study was to determine the pattern of change in the concentrations of 4 isoforms of tocopherol (α-, β-, γ-, and δ-tocopherol) in whole tissue and mitochondrial fraction from bovine liver and mammary gland after short-term feeding of a vegetable-derived oil (Tmix) particularly enriched with γ- and δ-tocopherol (9% α-, 1% β-, 24% δ-, and 62% γ-tocopherol). Nine healthy, multiparous Holstein cows (>90 d in milk) were assigned to dietary control (Notoc; n = 4) or tocopherol-fed treatment groups (n = 5; TOC; ~260 g Tmix/cow × d⁻¹, top-dressed) and fed for 9 consecutive days. On d 10 of feeding, tissues were harvested at slaughter and mitochondria were isolated. Tocopherol isoform concentrations were determined by HPLC and data were analyzed as a complete randomized design. Significance was declared at P ≤ 0.05. Concentrations of γ-tocopherol increased (0.01vs.0.07 µg/mg) in liver mitochondria from TOC cows compared with Notoc cows. In whole tissues and mitochondria, the α-isoform concentrations were higher than the γ-isoform. The accumulated portions of the α-isoform to the γ-isoform were similar for mitochondria and whole tissues regardless of tissue source differences. Regardless of tocopherol isoform or sample source (i.e., whole tissue vs. mitochondria), the liver accumulated higher total tocopherol concentration when compared with the mammary gland (8.2vs.2.7 µg/g, respectively). Limited concentrations of β- and δ-isoform were detected in whole tissues and mitochondria. In conclusion, the liver had higher tocopherol (α- and γ-isoforms) concentrations than the mammary gland suggesting that the liver may be preferred over the mammary gland for tocopherol accumulation. The α-isoform accumulated at higher concentrations than the γ-isoform in liver perhaps due to a higher affinity of tocopherol transport and binding proteins for α-tocopherol when compared with the γ-isoform.