Abstract: Objective: The fatty acid composition of peony seed oil for oil of different varieties and different origins in the Loess Plateau was studied, and the differences in peony seed oil for different oils were compared and evaluated. Methods: Gansu ‘Paeonia rockii’, Gansu, Inner Mongolia, Shanxi, Shaanxi ‘Fengdan’ peony was used as raw materials, peony seed oil was prepared, the oil content and basic physical and chemical indicators of peony seed oil were determined, and the composition of seed oil fatty acids was studied by two-dimensional gas chromatography and time-of-flight mass spectrometry (GC×GC-TOFMS). Results: The oil content of ‘Paeonia rockii’ and ‘Fengdan’ peony seed were 27.97~36.12 g/100 g, the saponification value of peony seed oil were 141~152 mg/g, the peroxide value were 1.24~1.48 mmol/kg, the refractive index were 1.465~1.467, and the color was light yellow and translucent. Peony seed oils contained a total of 17 fatty acids, more nervous acid and tetracosanoic acid were detected than the GC-MS results. The α-linolenic acid and oleic acid in ‘Paeonia rockii’ from Gansu were significantly higher than those in ‘Fengdan’ peony, but the linoleic acid was lower than that (P<0.05). There was the lowest oil content in Shaanxi ‘Fengdan’ peony, but it was better in terms of unsaturated fatty acid content and oleic acid and linoleic acid, its overall quality was lower than other varieties. With the increase of altitude and the decrease of precipitation, the oil content of peony seed oil for oil gradually increased. Conclusion: Peony seed oil for oil from different origins and varieties were different in terms of oil content, basic physical and chemical indicators and fatty acid content, and different growing conditions and varieties were the reasons for the differences. This study provided data support and theoretical support for the detection of fatty acid composition of peony seed oil for oil.