Rubus hirsutus Thunb., a typically red‐fruited shrub in the Rosaceae family, was found to include a stable yellow‐fruited variety at the Zhongshan Botanical Garden in Nanjing, China. This study provides the first comprehensive and multidimensional comparison of red (R) and yellow (Y) R. hirsutus fruits across three developmental stages (immature, color‐turning, and mature), integrating morphological, sensory, nutritional, and antioxidant analyses. For the first time, we systematically characterized sugar composition, amino acid profiles, phenolic compounds, and multiple antioxidant parameters (including total phenols, anthocyanins, glutathione, and antioxidant enzymes) in both color variants. Results revealed that mature yellow fruits exhibited significantly larger size (diameter and weight) than red fruits. While red fruits showed brighter coloration, higher soluble solid–acid ratio, and superior flavor scores, they accumulated significantly more fructose, total phenols, anthocyanins, trace elements (Fe and Zn), glutathione, and peroxidase activity at maturity. Conversely, yellow fruits contained higher levels of titratable acid (0.89% vs. 0.73%), total amino acids (2263.52 vs. 1835.68 μg/g in red), specific essential amino acids (His, Trp, and Ile), potassium, and a higher K/Na ratio. The protein content did not differ significantly between the phenotypes. Correlation analysis revealed that morphological indices were closely associated with sugar accumulation, whereas antioxidant indicators showed limited correlation with other parameters. These novel findings not only fill a critical gap in the characterization of yellow‐fruited Rubus germplasm but also provide a scientific foundation for targeted breeding and functional food development.

This study utilized mature fruits of red-fruited genotype ‘R’ of Rubus hirsutus Thunb and yellow-fruited form Rubus hirsutus Thunb. f. xanthocarpus (Nakai) M.Kim (‘Y’) as materials, employing HPLC, metabolomic, and transcriptomic techniques to describe the differences in amino acid metabolism, metabolite accumulation and gene expression between the mature fruits of the two genotypes of Rubus hirsutus, and analyze the metabolic characteristics between the yellow fruit variant and the wild red fruit. The results showed that the free amino acids of yellow-fruited ‘Y’ (1638.6 μg/g FW) were significantly higher than those of red-fruited ‘R’ (1331.8 μg/g FW), with glutamine showing the most significant difference. Metabolomics identified 232 differential metabolites: red fruits appeared dark red due to enrichment of anthocyanins, while yellow fruits exhibited bright yellow coloration dominated by chalcone glycosides and flavonols. Transcriptomic screening revealed 5052 differentially expressed genes, among which amino acid-related biosynthesis pathways showed the most significant differences. Specifically, genes encoding glutamine synthetase and those involved in branched-chain amino acid metabolism were highly expressed in ‘Y’ fruits, whereas genes associated with cysteine metabolism and aromatic amino acid metabolism were predominantly expressed in ‘R’ fruits. Collectively, these findings characterize the differences in amino acid metabolism, metabolites and transcripts between mature red-fruited and yellow-fruited Rubus hirsutus Thunb., and unravel the potential association between amino acid metabolism and fruit-color variation, providing a theoretical reference for subsequent research on the regulatory mechanisms governing fruit color and quality.