The aim of the work was to analyze the de-pendence of the total content of natural antioxi-dants (TCA) in the grain of Siberian barley varieties on genotype and growing conditions. Five varieties of spring filmy and hull-less barley grown during 2015 and 2016, in three different geographical lo-cations: Krasnoturansky, Beysky and Shirinsky state selection stations (SSS) were investigated. To determine TCA in the grain the samples were ex-tracted with two eluents - hot bidistilled water or 70 % ethyl alcohol. TCA measurement in the sam-ples was performed with the help of Yauza-01-AA color apparatus. Gallic acid was used as a refer-ence. The extraction methods used with water and alcohol showed similar results. Average value of the correlation coefficient between TCA, measured after extraction with water and after extraction with alcohol, was 0,974 ± 0,060. The maximum levels of TCA in the grain were noted in the barley grown in Beisk, and the minimum in Krasnoturansky GBS. The differences in TCA in grain over the years of barley cultivation were ambiguous. The tendency of the increase in TCA in barley grain was registered with the decrease in HTC value in August. It is sup-posed that more arid conditions for growing of bar-ley contributed to the accumulation of antioxidants in grain. Maximum levels of TCA in grain of barley were registered in grade Krasnoyarsk 91, and min-imum in the sample of Biome. When geographical location of barley growing was changed TCA in the grain changed, it led to the violation of ranking of varieties according to TCA.