Carotenoids are a diverse group of pigments imparting red, orange, and yellow hues to many horticultural plants, also enhancing their nutritional properties and health benefits. In strawberry, the genetic and molecular mechanisms regulating the natural variation of fruit carotenoid composition remain largely unexplored. In this study, we use a population segregating in yellow/white flesh to detect a major quantitative trait locus (QTL), qYellow Flesh-4B, located on chromosome 4B and accounting for 82% of total phenotypic variation. In the QTL interval, specific polymorphisms on the promoter of the carotenoid cleavage dioxygenase CCD4(4B) were associated with yellow flesh, down-regulation of CCD4(4B) during ripening, and increased carotenoid content. The role of CCD4(4B) in carotenoid turnover was further confirmed through transient overexpression in strawberry fruits, which resulted in decreased concentrations of the xanthophylls violaxanthin, lutein, and zeaxanthin. Notably, a -35 C>
T single-nucleotide polymorphism (SNP) in the CCD4(4B) promoter was predictive of both CCD4(4B) expression and carotenoid content across a diverse collection of octoploid Fragaria species. These findings provide valuable genetic insights into the natural variation of carotenoid composition and accumulation in strawberry. A high-resolution melting (HRM) DNA test developed in this study offers a rapid and reliable method for predicting high carotenoid content in strawberry fruits, representing a valuable tool for breeding projects aimed at enhancing the nutritional value of this crop.