Tomato (Solanum lycopersicum) is one of the most important crops used for human consumption worldwide since its fruit is a source of antioxidants and other bioactive compounds, very important for health and nutrition. Unfortunately, the erosion of genetic variability due to the domestication of tomato in most cases has led to the loss of valuable traits. The use of Introgression Line (IL) populations derived from a cross between the cultivated tomato (Solanum lycopersicum) and a wild species could help to restore important fruit quality traits into the genome of cultivated tomato. The Solanum pennellii IL population reconstitutes the wild type genome in 76 overlapping segments in the genetic background of S. lycopersicum (cv. M82). In a previous study, two S. pennelliii introgression lines (IL7-3 and IL12-4) harboring QTLs that regulate the synthesis of ascorbic acid (AsA) and phenolic compounds in the fruit were identified. The QTLs were pyramided into the genetic background of the cultivated tomato M82 using a marker-assisted selection approach. The cross between the two ILs produced different genotypes that were screened by species-specific markers in order to characterize their genomic constitution. The introgressed regions of S.pennellii in M82 were defined by exploring the Sol Genomics Network database (www.sgn.cornell.edu) in order to find polymorphic sequences between the reference genome S. lycopersicum (cv.Heinz) and S. pennellii, which allowed to design CAPS markers. A first molecular analysis was performed with six CAPS markers (three for region 7-3 and 12-4) positioned in the upper, middle and lower part of the introgressed regions. A further analysis with additional CAPS markers provided additional information about the length of the introgressed fragment in the sublines. Particularly, two sub-lines carrying the 7-3 region (analyzed with additional four CAPS markers) showed a wild type fragment of about 3.8 MB. Of the two sub-lines harboring the 12-4 region (analyzed with additional nine CAPS markers), the first sub-line showed about 2.1 MB of the wild type genome while a fragment of about 1.2 MB was found in the second sub-line. On the original introgression 7-3, a total number of 710 genes were mapped, whereas a total of 540 were mapped in the original introgression 12-4. In the selected sub-lines, a reduced number of about 620 genes for both IL 7-3A and IL-7-3B and of more than 300 for IL 12-4A and about 180 genes for 12- 4B were localized in the wild region. Consequently, phenotyping these sub-lines for AsA and phenolics could restrict the number of candidate genes to be further investigated. Preliminary biochemical analysis on tomato fruit of these sub-lines revealed different AsA and phenolics content comparing to the control genotype M82, indicating that genes controlling these metabolites are still present in the restricted wild regions. Further molecular and biochemical analyses will be carried out to better characterize the developed sub-lines. Moreover, these novel genotypes could be used in the future as genetic materials for a breeding scheme aimed at obtaining new hybrids or improved varieties with higher antioxidants levels.