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In root research, hydroponic plant cultivation is commonly used and soil experiments are rare. We investigated the response of 12‐day‐old barley roots, cultivated in soil‐filled rhizotrons, to different soil water potentials (SWP) comparing a modern cultivar (cv. Scarlett) with a wild accession ICB181243 from Pakistan. Water potentials were quantified in soils with different relative water contents. Root anatomy was studied using histochemistry and microscopy. Suberin and lignin amounts were quantified by analytical chemistry. Transcriptomic changes were observed by RNA‐sequencing. Compared with control with decreasing SWP, total root length decreased, the onset of endodermal suberization occurred much closer towards the root tips, amounts of suberin and lignin increased, and corresponding biosynthesis genes were upregulated in response to decreasing SWP. We conclude that decreasing water potentials enhanced root suberization and lignification, like osmotic stress experiments in hydroponic cultivation. However, in soil endodermal cell suberization was initiated very close towards the root tip, and root length as well as suberin amounts were about twofold higher compared with hydroponic cultivation. Summary Statement: In soil‐grown roots of barley, the endodermal cell suberization was initiated far much closer towards the root tip, suberin amounts were almost doubled, and roots were more than twofold longer compared with hydroponics. There is a very different plastic response to water stress in soil‐grown roots compared with hydroponically cultivated roots. [ABSTRACT FROM AUTHOR]