Abstract: This study aimed to investigate the effects of quality characteristics of different highland barley varieties on the glycemic index. A systematic comparison was conducted on the hardness, powder particle size, nutritional composition, gelatinization properties, and in vitro digestibility of 12 highland barley varieties. The influence of these factors on the estimated glycemic index (EGI) of highland barley was also analyzed. The results indicated that there was no consistent pattern in the nutritional composition among the different highland barley varieties. However, the hardness of the grains significantly influenced the grinding results; higher hardness typically led to larger particle sizes in the flour. The differences in the gelatinization properties of the highland barley flour from various varieties were not only associated with starch granule structure, starch content, and amylose content, but also with the variations in non-starch components present in the grains. Correlation analysis revealed varying degrees of association between EGI and the nutritional components of highland barley. EGI exhibited a significant negative correlation with amylose and β-glucan content (P≤0.05) and a certain degree of negative correlation with protein, polyphenols, powder particle size, and grain hardness. Through principal component analysis and cluster analysis, high-quality highland barley varieties with low EGI, high amylose, high β-glucan, and high protein content were identified: Chaiqing 1, Haixi Kunlun 15, Zangqing 3000, and Beiqing 8. These varieties had a comprehensive principal component score of 149.191 and an average EGI of 78.775. Ultimately, Zangqing 3000, with the best quality and the lowest EGI (75.51), was identified as the optimal variety. These findings provide an important theoretical basis for selecting and cultivating highland barley varieties in the Qinghai-Tibet region.