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Breeding of cereals with anthocyanin-rich grains is promising for health food production. Here, we transferred genes responsible for activation of anthocyanin biosynthesis in the grain pericarp (<i>Ant1</i> and <i>Ant2</i>) and aleurone (<i>HvMyc2</i>) to elite barley cultivars Aley, Tanay, and Vorsinsky-2 by marker-assisted backcrossing. Donors were Bowman lines BW648 and BW418. Three pairs of near-isogenic lines (NILs) with purple or blue colored grains were chosen in generation BC₆F₂ and propagated up to BC₆F₆. Genotyping-by-sequencing of resulting NILs and parental lines showed that the NILs carry 4.6–17.6% of donor SNPs including those at target loci. Unexpected big blocks of donor SNPs were revealed in some NILs on chromosomes 1H and 5H that do not carry target loci. The NILs possessed 274% and 12% higher anthocyanin (40.0–170.4 μg/g) and total phenolic content (2367.2–4396.8 μg/g), respectively, compared to original cultivars (18.3–33.1 μg/g and 2319.6–3768.5 μg/g). On average, antioxidant activity was 14% higher, although some lines showed reduced activity. NILs’ productivity depended on growth conditions and was similar to that of the recurrent cultivars. Hence, the applied breeding strategy is an effective approach to enrichment of grain anthocyanin content in barley.