We performed comparative metabolome and transcriptome analyses throughout fruit development using the tomato cultivar M82 and its near-isogenic line IL8-3, with interesting and useful traits such as a high content of soluble solids. Marked differences between M82 and IL8-3 were found not only in ripe fruits but also at 20 days after flowering (DAF) in the hierarchical clustering analysis of the metabolome, whereas patterns were similar between the two genotypes at 10 and 30 DAF. Our metabolome analysis conclusively showed that 20 DAF is an important stage of fruit metabolism and that the Solanum pennellii introgressed region in IL8-3 plays a key role in metabolic changes at this stage. Carbohydrate and amino acid metabolism were found to be promoted in IL8-3 at 20 DAF and the ripening stage, respectively, whereas transcriptome analysis showed no marked differences between the two genotypes, indicating that dynamic metabolic regulation at 20 DAF and the ripening stage was controlled by relatively few genes. The transcript levels of the cell wall invertase (LIN6) and sucrose synthase (TOMSSF) genes in starch and sucrose metabolic pathway and that of the glutamate synthase (SlGOGAT) gene in the amino acid metabolic pathway in IL8-3 fruit were higher than those in M82, and SlGOGAT expression was enhanced under high-sugar conditions. The results suggest that the promotion of carbohydrate metabolism by LIN6 and TOMSSF in IL8-3 fruit at 20 DAF affects SlGOGAT expression and amino acid accumulation via higher sugar concentration at the late stage of fruit development.