Previous reports showed that vitamin E in palm oil consists of various isomers of tocopherols and tocotrienols [α-tocopherol (α−T), α-tocotrienol, γ-tocopherol, γ-tocotrienol, and δ-tocotrienol), and this is normally analyzed using silica column HPLC with fluorescence detection. In this study, an HPLC-fluorescence method using a C₃₀ silica stationary phase was developed to separate and analyze the vitamin E isomers present in palm oil. In addition, an α-tocomonoenol (α−T₁) isomer was quantified and characterized by MS and NMR. α−T₁ constitutes about 3–4% (40±5 ppm) of vitamin E in crude palm oil (CPO) and is found in the phytonutrient concentrate (350±10 ppm) from palm oil, whereas its concentration in palm fiber oil (PFO) is about 11% (430±6 ppm). The relative content of each individual vitamin E isomer before and after interesterification/transesterification of CPO to CPO methyl esters, followed by vacuum distillation of CPO methyl esters to yield the residue, remained the same except for α−T and γ−T₃. Whereas α−T constitutes about 36% of the total vitamin E in CPO, it is present at a level of 10% in the phytonutrient concentrate. On the other hand, the composition of γ−T₃ increases from 31% in CPO to 60% in the phytonutrient concentrate. Vitamin is present at 1160±43 ppm, and its concentrations in PFO and the phytonutrient concentrate are 4,040±41 and 13,780±65 ppm, respectively. The separation and quantification of α−T₁ in palm oil will lead to more in-depth knowledge of the occurrence of vitamin E in palm oil.