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The potential of non-edible Reutealis trisperma oil as a new carbon source for biodiesel synthesis was investigated. The oil content in the kernels by hot pressing method was 50–52 wt%. The oil was processed into biodiesel directly without purification. The methanol concentration, reaction temperature, and the amount of catalyst used are crucially important to ensure high yield of biodiesel production. The optimisation of the synthesis condition achieved 95.15 % of biodiesel yield at 65 °C. The properties of biodiesel produced such as density; 887 kg/m³, flash point; 148 °C, acid number; 0.41 mg KOH/g, iodine number; 28.43 g-I²/100 g, total glycerol; 0.037 wt%, ester content; 97.18 wt%, pour point; −15 °C, and cetane number; 63 meet with ASTM standard D6751.