Abstract This study aimed to determine the variability of hexaploid wheat (Triticum aestivum), ancestral diploid wheat (T. monococcum) and rye (Secale cereale) root exudate potential to inhibit the arable weed black‐grass (Alopecurus myosuroides), in relation to variability in resistance to herbivorous pests and pathogens across the cereal germplasm. As benzoxazinoids are suggested to play a role in resistance against these stressors, and in allelopathy, we also aimed to identify compounds in root exudates. We conducted in vitro and glasshouse bioassays to determine the efficacy of a wide range of crude cereal root exudates and their constituent compounds in inhibiting black‐grass in both axenic and biologically‐active media. LC–MS analysis was used to characterise constituents of these exudates and differences between hexaploid wheat, diploid wheat and rye. Root development of black‐grass was suppressed to various degrees by crude root exudates from these cereals, with the most effective being S. cereale var. Edmondo and T. monococcum MDR037. Benzoxazinoid content of root exudates varied, with ancestral wheat lines and rye exuding fewer of these compounds than hexaploid wheat, but with higher variability between lines. Co‐culture with T. aestivum var. Gravity significantly inhibited early shoot growth and biomass of black‐grass seedlings, but individual benzoxazinoids had no effect on black‐grass in the same system. These data provide evidence that cereal–black‐grass interactions are influenced by root exudates, but that their effects cannot be replicated through direct application of individual constituent compounds.