This study uses PCR-derived marker systems to investigate the genetic differences of 22 grapevine accessions obtained through a self-fertilization program using Gaglioppo and Magliocco dolce. The aim of the study was to improve some qualitative parameters, while preserving the adaptive characteristics of these two cultivars to the adverse environmental conditions of the Calabria region (southern Italy). These two Calabrian grapevines have been cultivated within a restricted area and have been placed under a strong anthropic pressure which has limited their phenotypical variability with no selection of higher performant biotypes. Therefore, to have accessions with improved qualitative traits, a program of genetic improvement based on the self-fertilization of Gaglioppo and Magliocco dolce cultivars was performed in 1998, producing 3,122 accessions. Selection cycles were performed in 14 years. A first selection cycle (1998-2000), based on visual inspection of vegetative traits, selected 1,320 accessions, planted in an experimental vineyard in 2000. A second selection cycle (2000-2008), based on phenotypic traits, sanitary aspects, and chemical composition of the grapes, selected 42 accessions, planted in a new experimental vineyard in 2008. A final selection cycle (2008-2012), produced 22 accessions (virus free), with the best agronomic, sanitary, and qualitative aspects: two accessions obtained from Gaglioppo have been selected by color characteristics (i.e., anthocyanin total content and stability); 20 genotypes obtained from Magliocco dolce had a better macro-composition of the grape (i.e., good sugar content with a balanced acidity). SSR analyses were performed to check the self-fertilization process. The study of genetic differences between accessions was performed by AFLPs, SAMPLs, and M-AFLPs. The application of the above-mentioned techniques allowed both to discriminate molecularly the 22 accessions grouped these accessions according to their genetic similarity. The self-fertilization approach has enabled improvement in the quality of the grapes, while preserving the high degree of adaptation to the environment of these two native Calabrian cultivars in southern Italy.