Abstract This study investigates the effect of different crop rotation systems on carbon (C) and nitrogen (N) in root biomass as well as on soil organic carbon ( SOC ). Soils under spring barley and spring barley/pea mixture were sampled both in organic and conventional crop rotations. The amounts of root biomass and SOC in fine (250–253 μ m), medium (425–250 μ m) and coarse (>425 μ m) soil particulate organic matter ( POM ) were determined. Grain dry matter ( DM ) and the amount of N in harvested grain were also quantified. Organic systems with varying use of manure and catch crops had lower spring barley grain DM yield compared to those in conventional systems, whereas barley/pea showed no differences. The largest benefits were observed for grain N yields and grain DM yields for spring barley, where grain N yield was positively correlated with root N. The inclusion of catch crops in organic rotations resulted in higher root N and SOC (g C/m 2 ) in fine POM in soils under barley/pea. Our results suggest that manure application and inclusion of catch crops improve crop N supply and reduce the yield gap between conventional and organic rotations. The observed positive correlation between root N and grain N imply that management practices aimed at increasing grain N could also increase root N and thus enhance N supply for subsequent crops.