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Abstract: This study endeavored to investigate the variability of bacteria and fungi present during the fermentation process of the light-fragranced distilled liquor known as Fen liquor. To accomplish this, we used a combination of clone libraries of 16S rRNA genes, bar-coded pyrosequencing of the internal transcribed spacer region 1 (ITS1), and quantitative real-time PCR (qPCR). Fifteen families of bacteria and six families of fungi were detected. More than 91% of 16S rRNA gene sequences could be assigned to the family Lactobacillaceae, which were then classified to eight different operational taxonomic units (OTUs), based on a 3% cut-off. The most abundant OTU which contributed to 51% of the total 16S rRNA gene sequences was affiliated with Lactobacillus acetotolerans and had a significantly similar variation trend with the chemical constituents detected. Sixty percent of the fungal ITS1 region sequences were affiliated with the family Saccharomycetaceae. The most abundant OTU was very similar to Issatchenkia orientalis, which displayed notable similarities with respect to the change trends in both ethanol and organic acid contents. The sequences of the second most abundant OTU were closest to Saccharomyces cerevisiae, an important species in the process of ethanol production. Furthermore, about one fourth of the ITS1 region sequences belonged to the family Saccharomycopsidaceae. Conversely, very few sequences could be grouped together with filamentous fungi. The results of qPCR showed that the content of bacteria was increased while that of fungi was more stable in the fermentation process. It is very important to simultaneously investigate bacterial and fungal variations in food-fermentation processes. [Copyright &y& Elsevier]