Epidermal flavonoids have long been suggested as targets for selection in stress-adaptive crop breeding, but routine selection is neither practicable nor cost effective because of the time-consuming process involved in phenotyping. In this research, we found that the content of epidermal flavonoids, measured using the chlorophyll fluorescence excitation ratio method, was inversely related to stripe rust (Puccinia striiformis f. sp. tritici [Pst]) response in wheat (Triticum aestivum L.). We investigated the genetic basis by conducting quantitative trait locus (QTL) mapping with a 9K single nucleotide polymorphism (SNP) array in a cross of ‘Drysdale’ ´ ‘Waagan’ segregating for adult-plant stripe rust resistance. Data from 3 yr of field experimental trials were analyzed, and heritability estimates for epidermal flavonoids varied from 0.16 to 0.47 across the experiment-years. In contrast, heritability for stripe rust response varied from 0.79 to 0.84. Eleven QTL were identified for epidermal flavonoids, with a major locus on chromosome 4D, explaining between 19.5 and 47.3% of the phenotypic variation across years. Thirteen QTL were identified for stripe rust response, with the major locus explaining 68% of the phenotypic variation. Quantitative trait loci associated with epidermal flavonoids and stripe rust response were colocated on chromosomes 2B and 3B, but other genetic loci detected for the respective traits were independent. The study has established a genetic basis for epidermal flavonoids measured using the nondestructive, leaf-clip sensor in wheat, which will encourage further research on the possible exploitation of the compound by breeding and selection.