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Agricultural grass cultivars bred for increased forage yield commonly have extensive root systems. As roots are an important input of organic matter into the soil, it follows that such cultivars could lead to important increases in soil organic carbon (SOC), but this, and the mechanisms involved, are poorly understood with little empirical field evidence. We assessed the effect of two cultivars, Lolium perenne L. cv. "AberMagic" and L. perenne L. × Festuca pratensis Huds. cv. "Prior", in comparison with mixed‐species permanent pasture ("Pasture") in a field experiment on temperate alluvial soil in the southwest UK. Root and SOC stocks, and specific plant‐derived lipids as mechanistic biomarkers for shoot and root inputs of C, were measured on intact 1‐m volumetric soil cores collected on four dates in 2013–2015. Total grass yields were greater for both grass cultivars (40–43 Mg ha−1) than Pasture (39 Mg ha−1), and by 2015 root stocks in the upper 0.1 m were significantly greater by more than 4.7 Mg ha−1. At 0.1–0.3 m depth, SOC stocks (per 0.1‐m depth) were significantly greater under the two grass cultivars (24–50 Mg ha−1) compared to Pasture (18–32 Mg ha−1). In the full 1‐m profile, mean SOC stocks were 137, 133 and 110 Mg ha−1 for AberMagic, Prior and Pasture, respectively, matching root biomass distribution. Odd‐chain n‐alkane concentrations were significantly greater for shoots than roots (274 vs. 64 mg kg−1, respectively), and roots had significantly greater concentrations than shoots of even‐chain hydroxy fatty acids (1829 vs. 305 mg kg−1, respectively) and diacids (317 vs. 93 mg kg−1, respectively). This confirmed lipids as candidate biomarkers for distinguishing shoot and root inputs into the soil, with roots being the dominant source of SOC. Based on the observations of increased forage yield and SOC stock, high‐yielding forage grass cultivars can contribute to future strategies for climate‐smart agriculture in grassland‐dominated temperate regions. Highlights: Selected root traits for increased depth, density and diameter may increase SOC in grassland.The effect of two high‐yielding forage grass cultivars were assessed in a field trial.Fast‐growing, high‐yielding cultivars enhanced root and SOC stocks compared to typical pasture.Lipid biomarkers confirmed that root inputs were the predominant C input to soil. [ABSTRACT FROM AUTHOR]