Pot size is a critical factor in plant growth experiments, influencing root architecture, nutrient uptake, and overall plant development as well as sensing of stress. In controlled environments, variation in pot size can impact phenotypic and molecular outcomes and may bias experimental results. Here, we investigated how pot size affects the root system architecture and molecular responses of two barley genotypes, the landrace BERE and the modern elite CONCERTO, through assessment of shoot and root traits and by using X-ray computed tomography complemented by transcriptomic and metabolomic analyses. The two genotypes showed distinctly different adaptations to changes in pot size. The landrace showed greater stability and adaptability with consistent root traits and enhanced accumulation of osmoprotectant metabolites across different pot sizes with respect to CONCERTO. Conversely, the elite line was more sensitive to pot size variations, particularly showing altered root architecture and transcriptomic responses. Overall, this study highlights the importance of selecting an appropriate pot size for plant growth experiments, particularly when focused on root traits, and highlights the importance of considering the physiological and molecular changes due to growth environment choice in experimental design in barley.