Rhoifolin, a bioactive flavonoid with demonstrated anti-inflammatory and neuroprotective properties, represents an untapped opportunity for developing functional strawberries. We report extreme transgressive segregation (93-fold) for rhoifolin content amongst F1 progeny from cultivated strawberry (F. × ananassa) × Fragaria virginiana glauca crosses. LC-MS analysis revealed rhoifolin ranging from 2.1 to 195.1 million units, with genotype AN13,15,57 accumulating exceptional levels. Strong correlation between rhoifolin and rutin (ρ = 0.714, p = 0.058) validates the biological nature of this variation. Network analysis revealed coordinated elevation of 16 metabolites in high-rhoifolin genotypes, indicating systemic metabolic reprogramming rather than isolated changes. Distribution analysis suggests strong genetic control. This discovery opens immediate opportunities for marker-assisted selection of high-rhoifolin strawberries and demonstrates how wild germplasm unlocks hidden variation in cultivated fruit crops.