Chloroplast development and the chloroplast-to-chromoplast transition affect fruit development and quality. To further understand the effect of fruit coloration on fruit quality, tomato fruits with differentially colored shoulders were investigated at the microstructural, pigment, transcriptomic, and metabolic levels. Compared with fruits with a uniformly colored shoulder (US), chloroplast degradation was incomplete in green variegated shoulder I (VSI) fruits, and the number of plastoglobuli increased significantly in both VSI and yellow variegated shoulder II (VSII) fruits. Metabolite analysis showed that the contents of soluble solids, total organic acids, and vitamin C were significantly elevated in VSI fruits. In particular, sucrose accumulation in both VSI and VSII fruits increased to more than five times that of US fruits. Measurements of pigment contents in the fruit shoulder showed that both VSI and VSII fruits had significantly lower lycopene content than US fruits, whereas VSI fruits had higher chlorophyll content. Transcriptome analysis revealed that differentially expressed genes (DEGs) were significantly enriched in pathways related to sugar metabolism, carotenoid biosynthesis, lipid metabolism, and abiotic stress responses. In summary, the formation of variegated shoulders in tomato fruits was associated with defective chloroplast-to-chromoplast transition and altered pigment and metabolite accumulation.