The total flavonoid content, total polyphenols content and their antioxidant activities in eleven variety of hulless barley were determined and analyzed. Significant differences were found in active ingredients in hulless barley among different varieties and different habitats. The contents of total flavonoids and total polyphenols in Tibetan QB31, QB35 and Ni long hulless barley were significantly higher than those in eight kinds of Sichuan hulless barley, and the highest contents were 0.57 mg/g and 1.49 mg/g, respectively. Moreover, significant differences of total flavonoids content and total polyphenols content were detected in different varieties of hulless barley, and a strong positive correlation between total flavonoids content, total polyphenols content and antioxidant activity was found (P＜0.01). In addition, the clustering analysis of total flavonoids content and total polyphenols content could clearly distinguish Sichuan hulless barley from Tibet hulless barley, as well as dark hulless barley and light hulless barley.