Aims: The aim of this work was to obtain a deeper insight into the knowledge of microbial composition of Parmigiano Reggiano natural whey starters through different culture-independent methods. Methods and Results: Eighteen different Parmigiano Reggiano natural whey starters sampled from three different provinces of this cheese production area and the nonacidified wheys from which they arose have been studied by length heterogeneity polymerase chain reaction (LH-PCR) and fluorescent in situ hybridization (FISH). A high microbial composition variability between different samples has been observed. Conclusions: Revealing different images of the same community, LH-PCR and FISH have given a more accurate view of the not well-known Parmigiano Reggiano whey starter ecosystem. Significance and Impact of the Study: New lights have been shed on Parmigiano Reggiano natural whey starters microbial composition, highlighting how culture-independent approach could be used and improved to study this and other food ecosystems.