Barley is among the most ancient cereal crops, evolving over eons. The purposes of this study was to determine the total phenolic content, total flavonoids and total antioxidant capacity of barley grain collected from selected areas of Amhara region; and to compare it with other country barley and other cereal grains. A70% acetone in water was used to extract the phytochemicals. Total phenolic content was determined by Folin-Ciocalteu’s method, total flavonoids was determined by aluminium chloride methods while DPPH radical scavenging activity method was used to determine the total antioxidant activity and results were expressed as gallic acid equivalent), catechin equivalent and ascorbic acid equivalent respectively. Results of the determination revealed that total phenolic content, total flavonoids and total antioxidant capacity was ranging from 4.85±0.4 to 5.65±0.7 mg GAE/g, 3.45±0.4 to 4.01±0.7mg CE/g, and 2.98±0.8 to 3.69±0.4 mg AAE/g of sample respectively. Among the studied samples, sample from Derahamusite district exhibited a highest scavenging effect and the reducing capability on DPPH solution compared to samples from other district. Pearson correlation showed there were positive correlations between total phenolic content and total flavonoids content with antioxidant capacity. At 95% confidence level one way ANOVA displayed that the levels of total polyphenol, total flavonoid and antioxidant activity were not significantly varied between sampling sites. Furthermore, the magnitude of these bioactive chemicals are comparable amount with the other barley grain and other cereals reported in other literature.