The study used chlorophyll fluorescence imaging to map non-photochemical quenching (NPQ) gradients along barley leaf axes and found heat stress attenuates NPQ induction, revealing spatial heterogeneity in stress responses. Genome‑wide association and transcriptomic analyses identified candidate genes, notably HORVU.MOREX.r3.3HG0262630, that mediate region‑specific heat responses, highlighting pathways for improving cereal heat resilience.