The distribution of primary and secondary metabolites within a fruit can affect its nutritional and organoleptic quality, as fruit can vary in size and shape. This study investigated the differences in the distribution of the primary and secondary metabolites in the fruit of four strawberry cultivars (‘Asia’, ‘Clery’, ‘Frederica’, and ‘Sandra’) that were collected at one harvest point. The study included an analysis of the individual sugars, organic acids, phenolic compounds, and enzymes responsible for the degradation of phenolics. All of the studied cultivars showed a lower pH, higher total organic acid content, and lower glucose and fructose content in the outer part of the fruit. Differences were also observed in the total phenolic and anthocyanin contents, which were always higher in the outer part. The absolute differences in the total phenolic content ranged from 3723 to 6154 mg kg−1 dry weight. Our results provide a basis for understanding the differences in the biosynthesis of these metabolites within this fruit and prove that it is essential to mix samples well before extractions to obtain results that are representative of the whole fruit.
