In order to make better use of Tibetan hulless barley, the content of 17 amino acids (except tryptophan) for 72 hulless barley accessions was determined, and the nutritional value and flavor of amino acids was evaluated by WHO/FAO/UNU model. The cluster analysis was performed using the amino acid composition, and the difference of nutrition between landraces and modern varieties was surveyed. The total content of amino acids in hulless barley was 87.454 mg/g DW, with a range from 47.8 to 178.7 mg/g DW. The indispensable amino acid accounted for 37.15%, which was higher than that of oat, hulled barley and wheat. The Tibetan hulless barley exhibited the highest content (23.27%) of glutamine with large variation (CV=33%), and the lowest methionine (1.75%). The composition of amino acids in Tibetan hulless barley was similar to that in hulled barley and wheat. The Tibetan hulless barley had higher content of lysine compared with other cereal crop grains. The close degree U of hulless barley was 0.903, and 69.44% of the tested accessions was more than 0.900. That indicated that the composition of indispensable amino acid of Tibetan hulless barley were close to the model protein and thus had higher nutritional value. The Tibetan hulless barley genotypes presented broad variation on the sum of dispensable amino acids (28.7-118 mg/g DW), with the average of 55.785 mg/g DW. The content of amino acid contributing freshness was 26.58 mg/g DW and that for sweetness was 21.85mg/g DW. For nutrient balance, hulless barley was higher among cereal crops, the value of Score of Ratio Coefficient was 73.14. The content of amino acids in landraces/historic varieties was higher than that of improved varieties. Notably, four accessions were identified with the qualified nutritional value and flavor amino acid content. Thus, by unlocking the content of amino acids in hulless barley accessions, this study provided valuable insights to select elite genotypes in breeding for high-quality food hulless barley varieties.