Barley (Hordeum vulgare L.) grain is rich in fiber and antioxidant phytochemicals, including fatty acids, proteins, phenolic compounds, vitamins, and minerals, that offer various health benefits. Research on identifying different barley genotypes based on their health attributes is very limited. In this study, we performed an analysis of fatty acids, proteins, ascorbic acid, phenolic compounds, and antioxidant activity of several Canadian barley cultivars and elite breeding lines. Linoleic acid

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... (C18:2) was the predominant fatty acid present in the tested barley cultivars. The cultivar CDC Bold contained the highest amount of ascorbic acid, while the highest protein content was in CDC Mindon. An assessment of the free and bound phenolic compounds of barley grains showed quantitative changes among different genotypes of Canadian barley. Catechin is the most abundant molecule in free phenolics, while ferulic acid and para-coumeric acid are the most abundant in bound phenolics. Ferulic acid and vanillic acid were molecules detected in the soluble free fraction of all genotypes. Para-coumeric acid was detected only in genotypes such as CDC Copeland, CDC Bold, Lowe, and elite breeding Line 5 of both free and bound fractions of barley. Breeding Line 5 had the lowest antioxidant activity. An analysis of the above molecules and parameters of Canadian barley would help to uncover potential biomarkers in order to distinguish individual barley genotypes.