Abstract: To provide a scientific basis for the follow-up resource utilization and food development of Jinzhai hybrid G. elata, the ultra-high performance liquid chromatography coupled with mass spectrometry was utilized to accurately determine the contents of amino acids and nucleosides in Jinzhai Hongwu hybrid Gastrodia elata Bl. and its parents Yunnan G. elata Bl. f. glauca S. Chow and Jinzhai G. elata Bl. f. elata. Statistical methods were also applied to clarify and comprehensively evaluate the composition and content differences in amino acids and nucleosides among the three varieties of G. elata. C₁₈ column (2.1 mm×100 mm, 1.8 μm) was used with 0.2% formic acid water (A)-acetonitrile (B) as the mobile phase at a flow rate of 0.2 mL/min. The column temperature was 30 ℃ with gradient elution. Moreover, electrospray ion source was measured in multiple reaction monitoring (MRM) mode, which was set as spray voltage at 5500 V; ionization temperature at 550 ℃; ion source gas pressure (Gas) at 241.3 kPa, atomizing gas (GS1) at 379.2 kPa, and auxiliary gas (GS2) at 379.2 kPa. The results revealed that there was a good linear relationship in the range of 0~144.58 μg/mL of the 31 components. The coefficient of determination R² was greater than 0.999, and the RSD of the precision, stability, and repeatability of the peak area was not greater than 3.0%. The average recovery rate was between 92.69% and 99.5%, while recovery rate RSD ranged from 1.3% to 2.9%. Based on all above, G. elata was rich in amino acids and nucleotides, of which glutamic acid and aspartic acid were the main components. The contents of total amino acids and nucleosides in hybrid G. elata were higher than those of its parents G. elata. And there was a significant difference in the amino acids contents of different varieties (P<0.05). In the evaluation results of PCA and TOPSIS methods, the qualities of S1 and S2 hybrid G. elata were the best. This paper established a new method for evaluating the quality of G. elata and provided a scientific basis for the development and exploitation of Jinzhai hybrid varieties.