The genetic diversity of tomato in Italy and the growing interest in high-quality food products highlight the importance of establishing varietal distinctiveness through molecular strategies to ensure agrifood product quality and traceability. In this study, four Italian potato-like leaf (PL) landraces were analyzed: “Spagnoletta di Formia e di Gaeta” (SPA) from southern Lazio, “Giagiù” (GIA) and “Patanara” (PTN) from Campania, and “Pomodoro di Mola” (MOL) from Apulia. These landraces were genotyped for the potato leaf gene (C), with two PL American genotypes and a non-allelic PL mutant line included as outgroups. Nagcarlang served as control. An allelism test confirmed C as determinant gene. The SCAR marker for C revealed that the Italian landraces presented determinants other than the most representative one responsible for PL. Whole-genome sequencing of SPA identified a private novel nonsense SNP variant allele, confirmed through dCAPS marker analysis. Additionally, two novel PL alleles responsible for missense variations were identified in GIA/PTN and MOL. In silico protein analysis suggested that novel C alleles could be functional determinants for the protein activity. Overall, PL mutations identified for the first time could serve as molecular tools for agrifood chain traceability, enabling early differentiation and recognition of genotypically similar varieties.