To assess the potential of traditional selection breeding to develop varieties with increased phytosterol content, we determined concentrations of those sterols in canola, sunflower, and soybean seed oils produced from breeding lines of diverse genetic backgrounds. Seed oils were extracted and saponified, and the nonsaponifiable fractions were subjected to silylation. The major phytosterols brassicasterol, campesterol, stigmasterol and β-sitosterol, were quantified by capillary gas chromatography with flame-ionization detection. Canola contained approximately twice the amount of total phytosterols (4590–8070 μg g−1) as sunflower (2100–4540 μg g−1) or soybean (2340–4660 μg g−1) oils. Phytosterol composition varied among crops as expected, as well as within a crop. Both genetic background and planting location significantly affected total phytosterol concentrations. Soybean plants were maintained from flower initiation to seed maturity under three temperature regimes in growth chambers to determine the effect of temperature during this period on seed oil phytosterol levels. A 2.5-fold variability in total phytosterol content was measured in these oils (3210–7920 μg g−1). Total phytosterol levels increased with higher temperatures. Composition also changed, with greater percent campesterol and lower percent stigmasterol and β-sitosterol at higher temperatures. In these soybean oils, total phytosterol accumulation was correlated inversely with total tocopherol levels. Owing to the relatively limited variability in phytosterol levels in seed oils produced under field conditions, it is unlikely that a traditional breeding approach would lead to a dramatic increase in phytosterol content or modified phytosterol composition.