The Solanum pennellii introgression lines (ILs) have been exploited to map quantitative trait loci (QTLs) and identify favorable alleles that could improve fruit quality traits in tomato varieties. Over the past few years, ILs exhibiting increased content of ascorbic acid in the fruit have been selected, among which the sub-line R182. The aims of this work were to identify the genes of the wild donor S. pennellii harbored by the sub-line and to detect genes controlling ascorbic acid accumulation by using genomics tools. A Genotyping-By-Sequencing (GBS) approach confirmed that no wild introgressions were present in the sub-line besides one region on chromosome 7. By using a dense single nucleotide polymorphism (SNP) map obtained by RNA sequencing (RNA-Seq), the wild region of the sub-line was finely identified; thus, defining 39 wild genes that replaced 33 genes of the ILs genetic background (cv. M82). The differentially expressed genes mapping in the region and the variants detected among the cultivated and the wild alleles evidenced the potential role of the novel genes present in the wild region. Interestingly, one upregulated gene, annotated as a major facilitator superfamily protein, showed a novel structure in R182, with respect to the parental lines. These genes will be further investigated using gene editing strategies.