Lack of O2 and high concentrations of iron (Fe) are common in flooded soils where Rice (Oryza sativa L.) is cultivated. We tested the hypothesis that growing in stagnant or high Fe conditions might induce the formation of apoplastic barriers in roots with different properties and chemical compositions. We measured radial O2 loss (ROL) from intact roots, the chemical composition of roots and the formation of root iron plaques in Fe‐sensitive and Fe‐tolerant rice genotypes grown in aerated, deoxygenated stagnant solutions or in aerated solutions containing high concentrations of Fe. Roots of plants grown in stagnant conditions developed tight barriers to ROL, while those grown in high Fe conditions developed only partial barriers. Chemical analysis of enzymatically isolated sclerenchyma/exodermal root cells indicated that roots grown in stagnant conditions showed increased amounts of suberin and glycerol esters. In contrast, roots in high Fe conditions showed an increase in suberization along with formation of iron plaques covering the roots. In high Fe conditions, exodermal suberization and the formation of partial ROL barriers were not influenced by the genotype's tolerance to Fe. The amount of O2 diffused from roots is influenced by the various layers that impede O2 diffusion. Specifically, increased amounts of glycerol esters in the suberized exodermis provide the greater resistance to ROL.