Abstract: To explore the differences in the composition, content and comprehensive quality of free amino acids (FAA) in millet from Longzhong area in Gansu province, 12 different cultivars of millet were used as research objects, the types and contents of FAA were determined by liquid chromatography-tandem mass spectrometry. Comprehensive evaluation of FAA quality was performed by principal component analysis (PCA) and hierarchical cluster analysis (HCA). The results showed that 19~20 kinds of FAA were detected in all kinds of millet, and the total content of FAA ranged from 1046.80~1773.85 mg/kg, asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu) and glycine (Gly) were the four main FAA in millet. There were significant differences in the contents of FAA (P<0.05), but no obvious difference in species in the 12 cultivars of millet. The characteristics of flavor amino acids in millet were delicious amino acids>bitter amino acids>sweet amino acids>aromatic amino acids. 9 medicinal amino acids and 3 branched chain amino acids were found in millet, the average content of medicinal amino acids was 706.34 mg/kg, the percentages of medicinal amino acids to total FAA was 53.63%, so millet had many potential medicinal values. There was a significant relation with FAA in different cultivars of millet, five principal components were extracted after PCA, and the cumulative contribution ratio of the five components was 86.20%, the FAA comprehensive scores of LG-23、LG-11 and YG-18 were ranked as the top three. The 12 different cultivars millet were divided into four groups by HCA, FAA comprehensive quality performances of groups Ⅰ (Longgu-23) and III (Longgu-11 and Yugu-18) were optimal, the content of total FAA was significantly higher than the other two groups (P<0.05), but group Ⅱ (Lianggu from Huining、Longgu-18、Longgu-029) had relatively poor FAA quality, and the results were consistent with the PCA, which reflected the distinction among different cultivars of millet．