Abstract: This paper aimed to explore the biochemical characteristics of fresh leaf raw materials of large-leaf cultivars. In this study, four different tenderness fresh tea leaves of single bud, one bud with one leaf, one bud with two leaves and one bud with three leaves picked from Yunnan large-leaf cultivars (Qingshui No.3, Xianggui Yinhao, Yunkang No.10, Yunkang No.14, Yuncha Purui) were selected as the research objects. The variation of raw material taste quality with tenderness of different varieties of Yunnan large-leaf cultivars was investigated by detecting the content of its main flavor substances (tea polyphenols, flavonoids, amino acids and soluble sugars, etc.). Multivariate statistical methods were used to explore the categories and key differential indexes of fresh leaves with different tenderness, meanwhile, fisher discriminant analysis was used to establish the discriminant models for different types of fresh leaves. The results showed that the higher the maturity of fresh leaves, the higher values of phenol ammonia ratio, simple catechins, flavonoid glycosides and peroxidase would be, and one bud with two leaves and one bud with three leaves were significantly higher than single bud and one bud with one leaf (P<0.05). The higher the maturity of fresh leaves, the lower values of a gallic acid and amino acid would be, and single bud and one bud with one leaf were significantly higher than one bud with two leaves and one bud with three leaves (P<0.05). With the decreasing of tenderness, the values of tea polyphenols, ester catechins and caffeine increased first and then decreased, and compared with one bud with three leaves, the contents of tea polyphenols, ester catechins and caffeine in one bud with two leaves increased by 7.67%, 13.77% and 10.22%, respectively. The components with higher content of flavonoids and soluble sugars were rutin and sucrose, respectively, and its content ranged from 0.03% to 0.93%, 0.14% to 1.14%, respectively. The Yunkang No.14 with single bud and Qingshui No.3 with one bud with two leaves respectively had better material basis suitable for processing green tea and black tea. The four different tenderness could be divided into two types, that was, single bud and one bud with one leaf was a group, one bud with two leaves and one bud with three leaves was another group, and simple catechins, phenol ammonia ratio, peroxidase and flavonoid glycosides were the key difference indicators between the two kinds of fresh leaves. The accuracy rate of Fisher discriminant model for fresh leaf classification was 85%. This study would provide a theoretical reference for the tea shoots classification and standardized processing of Yunnan species.