The quality and yield of Luzhou-flavor liquor is directly determined by the quality of pit mud from the Luzhou-flavor liquor distillery. The aged pit mud produces the good quality liquor while new pit mud dose not. The aim of this study was to investigate the prokaryotic community diversity between new and aged pit mud by high-throughput sequencing technique and the real-time quantitative PCR (qPCR) assay. Results indicated that diversities of prokaryotic community in new pit muds were higher than those in aged pit muds. The abundances of the phylum Bacteroidetes in aged pit muds were significantly (P < 0.05) higher than those in new pit muds, while the abundances of the genus Lactobacillus and Bacillus changed in the opposite trend. Additionally, the quantity of Clostridium Kluyveri in aged pit mud was higher than that in new pit mud. Bacteroidetes, Lactobacillus, Bacillus and C. kluyveri may be the potential indicator-like microbe to distinguish new and aged pit mud fast, conveniently and reliably by qPCR. Results presented in this study provide specific understanding of the differences in prokaryotic community between the new and aged pit muds, and have laid initial foundation for identifying aged pit mud and for advancing pit mud aged.