The influence of internal (genetic and developmental) and external (environmental) factors on levels of flavonoid gene transcripts, enzyme activity and metabolites was studied in fruit of six cultivated strawberry (Fragaria x ananassa Duch.) genotypes grown at two Italian locations. Gene expression and enzyme activity showed development- and genotype-associated patterns, revealing gene coordination. Analysis clarified the regulation mechanism of the hydroxylation status of the B-ring of the major flavonoid pools and pointed out examples of genotype-specific post-transcriptional regulation mechanisms and key steps of pathway regulation in strawberry fruits. Metabolite profiles were strongly affected by development and genotype. Flavan-3-ols, their proanthocyanidin (PA) derivatives and anthocyanins were the most abundant metabolites. Flavonol levels and PA-associated traits (epicatechin/catechin ratio and mean degree of polymerization) showed significant environmental effects. Multivariate and correlation analyses determined the relationships among genes, enzymes and metabolites. The combined molecular and biochemical information elucidated more in depth the role of genetic and environmental factors on flavonoid metabolism during strawberry fruit development, highlighting the major impact of developmental processes, and revealing genotype-dependent differences and environmental effects on PA-related traits.