High-temperature Daqu is crucial to Jiang-flavor Baijiu production in the Southern Sichuan–Northern Guizhou region of China. However, the complex interplay among microorganisms, enzymes, and metabolites in the Daqu from this region requires further investigation. This study compared four high-temperature Daqu samples from this region, analyzing their physicochemical properties, enzyme activities, volatile compounds, and microbial community composition and function, and exploring the influence of microorganisms on the saccharification and aroma-formation function of Daqu in combination with correlation analysis. The microbial communities in the Daqu samples exhibited functional redundancy, with Desmospora sp. 8437 being consistently dominant (3.6–7.3%). Members of the family Bacillaceae were the principal factors contributing to the differences in starch degradation capacity, protein degradation capacity, and pyrazine content among the Daqu samples, mainly through the amylases and proteases they produce. Kroppenstedtia spp. were principal factors causing the differences in aldehyde and ketone contents, primarily via the lipid degradation enzymes they synthesize. Overall, the bacterial community composition of Daqu greatly influenced its characteristics. This study provided a theoretical basis for understanding the diversity of high-temperature Daqu in the Southern Sichuan–Northern Guizhou region.

High-temperature Daqu (HTD)’s quality determines the characteristics and yield of the Chinese sauce-aroma baijiu. However, winter production frequently encounters challenges such as fermentation instability and metabolic fluctuations, primarily stemming from complex, unmonitored microenvironmental changes within the HTD pile. This study established a closed-loop system linking the microenvironment, HTD quality, microbiome, and metabolome. Through continuous monitoring of the winter fermentation pile’s microenvironmental conditions and integrating multi-omics analyses, we revealed that CO₂ concentration within fermentation piles is the core factor causing quality variations in HTD. By breaking the respiratory bottleneck formed by carbon dioxide (CO₂) accumulation through the turning anaerobic stress can be alleviated, thereby driving metabolic succession. The study found that vertical CO₂ concentration heterogeneity severely restricts the enrichment of aerobic core functional microbial communities such as the Bacillus species. This directly blocks key metabolic pathways including amino acid metabolism and energy supply via ABC transporters. Moreover, the specific accumulation of Amadori products further confirms that this low-temperature environment under CO₂ stress causes the Maillard reaction to stall at intermediate stages. Consequently, this study proposes a steady-state control strategy centered on oxygen and CO₂ gas characteristics. By actively regulating the gaseous microenvironment to eliminate metabolic heterogeneity, it provides theoretical support for standardizing traditional fermentation processes.