Abstract:[Background] Vinegar Daqu is an important source of microorganisms during the brewing of traditional grain vinegar in China. It is usually prepared once and used in batches. [Objective] To reveal the structural variations of the microbial community in the traditional vinegar Daqu during the long-term storage. [Methods] From a century-old vinegar factory in southern Shanxi, we collected the raw materials of Daqu and the vinegar Daqu products that were newly produced and had been stored for 7 and 12 months, respectively. Microbial diversity was investigated by high-throughput DNA sequencing. [Results] A total of 610 fungal operational taxonomic units (OTUs) and 747 bacterial OTUs were obtained from the four groups of samples. Ascomycota (95%) and Firmicutes (81%) were dominant among fungi and bacteria, respectively. About one third of fungal OTUs and 95% of bacterial OTUs in vinegar Daqu were detected in the raw materials of Daqu, indicating that raw materials acted as the main source of microorganisms in vinegar Daqu. Compared with that in the newly-produced vinegar Daqu, the diversity of fungi and bacteria decreased significantly in vinegar Daqu samples that have been stored for 7 months and 12 months. Microbial community structure changed during the storage of vinegar Daqu, with the bacterial community structure being more prone to fluctuation than the fungal community. Compared with those in the raw materials, fungi like Saccharomycopsis fibuligera and Issatchenkia orientalis and bacteria such as Kroppenstedtia were significantly enriched in vinegar Daqu products. According to the results of function prediction, the fungi were mainly saprotrophic, and the metabolism pathways of bacteria were enriched significantly. [Conclusion] Vinegar Daqu presented decreased microbial diversity and varied microbial community structure during the long-term storage. This study enriched our understanding of the microorganism source in traditional vinegar Daqu and the variation patterns of microbial community structure in vinegar Daqu during storage, providing a scientific basis for stabilizing Daqu quality and improving the production process.