The objective of this study was to determine the pattern of change in the concentrations of the 4 isoforms of vitamin E (α-, β-, γ- and δ-tocopherol) in bovine tissues (liver, mammary and muscle), blood and milk after short-term feeding of a vegetable-derived oil (Tmix) particularly enriched with γ- and δ-isoforms (9% α-, 1% β-, 24% δ- and 62% γ-tocopherol). Healthy multiparous Holstein cows (>90 DIM) were assigned to dietary control (Notoc, n = 4) or tocopherol-fed treatment groups (n = 5: TOC;~260g Tmix/cow/d, top-dressed) for 9 consecutive days. Milk and blood samples were collected in the mornings on d 0 and d 10 from the 5 TOC cows; tissues were harvested from those same cows on d 10 of treatment and the cohort of physiologically similar Notoc cows. Isoform concentrations were determined in samples by HPLC. Data were analyzed by Student’s t-test and ANOVA. Delta-TOC was not detected in tissues, blood or milk. Concentrations of γ-TOC were increased (P < 0.001) in tissues, blood and milk with feeding (P < 0.001). In all tissues, α-TOC concentrations were higher (P < 0.001) than γ-TOC before and after feeding. Concentrations of both α- and γ-TOC were higher in blood and milk after feeding Tmix (P < 0.05). The content of α-TOC was higher than γ-TOC (P < 0.001) in milk and blood before and after feeding. In conclusion, short-term feeding of Tmix oil resulted in increases in the γ-isoform, largely no measurable content or significant change in the β- or δ-isoforms, and a variable change in the α-isoform of TOC across various tissues and biological fluids of the cow.