Aims: Verify the presence and the molecular identity of phytoplasmas in Northern and Central Italy vineyards where yellows diseases are widespread. Methods and Results: Phytoplasma presence and identity were determined by PCR/RFLP analyses on 16S ribosomal gene testing 1424 symptomatic samples. The 65% of samples resulted phytoplasma infected; in particular 256 samples were found positive to phytoplasmas belonging to group 16SrV (mainly Flavescence doree associated), and the remaining 37% was infected by phytoplasmas belonging to ribosomal subgroup 16SrXII-A (Stolbur or Bois Noir associated). 16SrV ribosomal group representative strains were further typed for variability in SecY and rpS3 genes. The results showed the presence of phytoplasmas belonging to 16SrV-C, 16SrV-D and to a lesser extent, 16SrV-A subgroup. Conclusions: Possible relationships between genetic polymorphisms of phytoplasma strains belonging to subgroup 16SrV-C and their geographic distribution and/or epidemic situations were detected. Significance and Impact of the Study: Bois Noir and Flavescence doree phytoplasmas are present in significant percentages in the areas under investigation. Molecular tools allowed to identify phytoplasma-infected plants and the genes employed as polymorphism markers resulted useful in distinguishing and monitoring the spreading of the diseases associated with diverse phytoplasmas belonging to 16SrV subgroup in vineyards.