The genetic grapevine intravarietal variability will be analyzed by PCR-derived marker systems. In particular, the object of the investigation will be the clonal variations of Malvasia nera di Brindisi/Lecce, Negroamaro and Primitivo, also known as Zinfandel, which are three grapevine varieties cultivated in Apulia region (Italy). In order to assess varietal identity of the samples, 132 DNA tests were performed by amplifying 16 SSR loci. The study of the intravarietal variability was performed using AFLPs, SAMPLs, ISSRs, and M-AFLPs. The application of the above-mentioned techniques allowed both to discriminate all genotypes of the three cultivars and to distinguish the accessions of each cultivar sampled from different geographic cultivation areas. Furthermore, the study of biotypes cultivated in different geographical environments of Salento (i.e., Apulia region) allowed important correlations between molecular marker variability and phenotypic traits. These results are suggesting both to focus our attention on the effects of the environment on the genotype and to consider, as a practical consequence, the importance of preserving autochthon grapevine biotypes found in different areas to truly preserve the richness of the germplasm. Thus, more accurate DNA studies give new information that can be extremely useful to the vine nurseries for the correct choice (i.e., supported by more accurate intravarietal variability analysis) of the grape multiplication materials.