Objective: This study aimed to provide the basis for selection and identification, quality breeding and comprehensive processing and utilization of millet germplasm resources. Methods: 12 varieties of millet were used to be tested objects, amino acid content of millet were measured, and then the content of 17 kinds of amino acids in millet were used to be evaluation index and applied to principal component analysis and comprehensive evaluation of quality. Millet varieties were clustered by clustering analysis. Results: The affected order of millet varieties on the content of 17 kinds of amino acids was Ser＞Lys＞Arg＞His＞Met＞Ile＞Thr＞Gly＞Pro＞Tyr＞Phe＞Asp＞Cys＞Glu＞Leu＞Val＞Ala. 2 principal components were extracted by principal component analysis, and the accumulative variance contribution rate reached 88.329%. Glu, Pro, Leu, Phe, Ile, Lys, Thr, His and Tyr could be used to comprehensive assessment index of millet varieties. Quality merits order of the 12 millet varieties were obtained by subordinate function methods, and they could be divided into three groups by clustering. Conclusion: Comprehensive quality of Jingu61 is the best, and that of Zhangzagu13 is the worst.