A barrier to radial O2 loss (ROL) in roots is an adaptive trait of waterlogging-tolerant plants. Hordeum marinum Huds. is a waterlogging-tolerant species that, in contrast to its waterlogging-sensitive cultivated relatives, forms a tight barrier to ROL in basal root zones. To evaluate the nature of the barrier to ROL in roots, we combined measurements of ROL with histochemical and biochemical studies of two contrasting H. marinum accessions. H21 formed greater aerenchyma (up to 38% of cross-sectional area) and a tight barrier to ROL when grown under deoxygenated stagnant conditions, whereas the barrier was only partially formed in roots of H90 and aerenchyma was up to 26%. A tight barrier to ROL in roots of H21 corresponded with strong suberisation of the exodermis. In agreement with anatomical studies, almost all aliphatic suberin quantities were greater in roots of H21 grown under stagnant conditions compared with roots from aerated controls, and also to those in H90. By contrast to suberin, no differences in root lignification were observed between the two accessions raised in either aerated or stagnant conditions. These findings show that in adventitious roots of H. marinum, suberisation rather than lignification restricts ROL from the basal root zones.