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Tomato (Solanum lycopersicum) is a crucial component of the hu- man diet because of its high nutritional value and the antioxidant content of its fruit. As a member of the Solanaceae family, it is considered a model species for genomic studies in this family, especially since its genome has been completely sequenced. Among genomic resources available, Solanum pennellii introgres- sion lines represent a valuable tool to mine the genetic diversity present in wild species. One introgression line, IL12-4, was previously selected for high ascorbic acid (AsA) content, and a transcriptomic analysis indicated the involvement of genes control- ling pectin degradation in AsA accumulation. In this study the integration of data from different “omics” platforms has been ex- ploited to identify candidate genes that increase AsA belonging to the wild region 12-4. Thirty-two genes potentially involved in pathways controlling AsA levels were analyzed with bioinformatic tools. Two hundred-fifty nonsynonymous polymorphisms were detected in their coding regions, and 11.6% revealed deleterious effects on predicted protein function. To reduce the number of genes that had to be functionally validated, introgression sublines of the region 12–4 were selected using species-specific polymor- phic markers between the two Solanum species. Four sublines were obtained and we demonstrated that a subregion of around 1 Mbp includes 12 candidate genes potentially involved in AsA accumulation. Among these, only five exhibited structural delete- rious variants, and one of the 12 was differentially expressed between the two Solanum species. We have highlighted the role of three polymorphic pectinesterases and inhibitors of pectinester- ases that merit further investigation.