The effect of cultivar on sterol composition of tomato (Solanum lycopersicum L.) seed oil from three industrial cultivars (Principe Borghese, Rebelion F1 and San Marzano) grown in Calabria Region (Southern Italy) was investigated. Sterol composition is presented both as percentage on the total sterol content (%) and as an absolute value (mg/kg). Rebelion F1 produced the worst seed oil in terms of cholesterol content (15.76%, 275.90 mg/kg). Principe Borghese produced the oil with the highest β-sitosterol (57.47%, 1088.33 mg/kg) and the highest apparent β-sitosterol (67.59%, 1279.62 mg/kg). One-way ANOVA and PCA analyses demonstrated a high and significant difference among the seed oils of the studied tomato cultivars.