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Higher alcohols are not only the key flavor substance but also the precursor of other flavor substances in Baijiu. In the metabolic pathway of higher alcohols, alcohol dehydrogenase (ADH) plays an important role and its genes can be used as a marker gene for analyzing the producer of higher alcohols. In this study, the degenerate primers of Zinc-dependent long-chain ADH gene (L.adh) were designed and applied to investigate the diversity of L.adh in different Daqu (HT, SC and SD). The results showed that the degenerate primers were able to amplify the fragment of L.adh and had good coverage in high-throughput sequencing. The diversity and composition of L.adh in the three kinds of Daqu were different and the diversity of L.adh in SC was the highest while HT was the lowest. The predominant L.adh in SC and HT were form Bacillus (54%–78%) and Aspergillus (64%–66%), respectively, and the predominant L.adh in SD were from Aspergillus (7%–51%) and Enterobacter (2%–47%). In addition, L.adh from bacteria and fungi could be clustered into one evolutionary branch and divided into 10 clusters. The findings of this study will provide a new perspective for evaluating the potential microorganisms associated with higher alcohol synthesis in Daqu and reference for selecting suitable Daqu in the production of Baijiu.