Oil derived from different lines of genetically modified canola varieties was analyzed for phospholipids, tocopherols, and phytosterols by various chromatographic techniques. As observed previously in genetically modified soybean oils, there was a decrease in the content and composition of phosphatidic acid in three of the modified canola oils derived from the 12 varieties investigated. Normal-phase high-performance liquid chromatographic (HPLC) analyses showed small variations in the phospholipid content of major classes, despite few differences in their composition. Reversed-phase HPLC data indicated that the molecular species distribution of phosphatidylethanolamine was significantly altered by genetic modification when compared to phosphatidylcholine. Impact of oilseed modification on the tocopherol content was variable, with greater variation in the concentration of α- and γ-tocopherols than δ-tocopherol. Phytosterol composition was markedly affected by genetic modification. Brassicasterol, campesterol, and β-sitosterol levels were consistently lowered in one genotype, whereas increased brassicasterol content was observed in the other variety. In general, genetic modification of canola seeds led to changes in the distribution of phospholipids, tocopherols, and phytosterols.