The aim of this paper is to explore the amino acid composition and nutritional evaluation of mare milk and traditional fermented mare milk in two pasture areas of western China. In this study, 40 samples of mare milk and traditional fermented mare milk were collected in the main pastoral areas of Xinjiang and Inner Mongolia. An amino acid automatic analyzer and high performance liquid chromatography were used to determine the composition and content of free amino acids and hydrolyzed amino acids in the samples. The obtained data were analyzed using analysis of variance (ANOVA) and nutritional evaluation was performed. The results showed that there was no significant difference in the total amino acid of mare milk in both areas (P > 0.05); the free amino acid contents of two fermented mare milk samples were generally significantly increased relative to their unfermented counterparts (P < 0.05), and new amino acids with special physiological activities were produced such as gamma-aminobutyric acid (GABA), beta-aminoisobutyric acid (BAIBA) and gammahydroxylysine; the content of sweet amino acid Xinjiang mare milk was significantly increased after fermentation, and the total amino acid content was higher than in fresh mare milk, indicating that fermentation helped improve the flavor of mare milk; the essential amino acid index values of fresh and fermented mare milk from the two areas were all greater than 95, indicating that both fresh and fermented mare milk were high-quality protein sources. Moreover, the nutritional evaluation of fermented mare milk was higher than fresh mare milk in each area.