Mozzarella (M), Grana Padano (GP) and Parmigiano Reggiano (PR) are three of the most important traditional Italian cheeses. In the three cheese manufactures the initial fermentation is carried out by adding natural whey cultures (NWCs) according to a back-slopping procedure. In this study, NWCs and the corresponding curds from M, GP and PR manufactures were analyzed by culture-independent pyrosequencing of the amplified V1-V3 regions of the 16S rRNA gene, in order to provide insights into the microbiota involved in the curd acidification. Moreover, culture-independent high-throughput sequencing of lacS gene amplicons was carried out to evaluate the biodiversity occurring within the S. thermophilus species. Beta diversity analysis showed a species-based differentiation between GP-PR and M manufactures indicating differences between the preparations. Nevertheless, all the samples shared a naturally-selected core microbiome, that is involved in the curd acidification. Type-level variability within S. thermophilus species was also found and twenty-eight lacS gene sequence types were identified. Although lacS gene did not prove variable enough within S. thermophilus species to be used for quantitative biotype monitoring, the possibility of using non rRNA targets for quantitative biotype identification in food was highlighted.