Abstract: In this study, PacBio SMRT sequencing technology and conventional physicochemical analysis methods were employed to conduct a comparative analysis of the fungal community structure and physicochemical characteristics of medium-temperature Daqu samples from Luzhou and Nanyang regions. Furthermore, principal coordinate analysis (PCoA) and correlation analysis were performed to reveal the association between dominant fungal species and physicochemical properties. The results of fungal community analysis showed that the fungal communities in medium-temperature Daqu from both Luzhou and Nanyang regions were dominated by Thermoascus aurantiacus, Thermomyces lanuginosus, and Saccharomycopsis fibuligera, with cumulative average relative abundances of 64.59% and 83.58%, respectively. Additionally, Rasamsonia composticola and Issatchenkia orientalis were identified as fungal biomarkers for medium-temperature Daqu from Luzhou and Nanyang regions, respectively. Physicochemical analysis results indicated that, compared to medium-temperature Daqu from Nanyang, Daqu from Luzhou exhibited significantly higher water activity, moisture content, bound water content, ash content, starch content, amino acid nitrogen content, and protein content (P<0.05), while density, crust hardness, free water content, liquefaction power, esterification power, and saccharification power were significantly lower (P<0.05). Correlation analysis revealed complex relationships between dominant fungal species and physicochemical indicators in medium-temperature Daqu. This study provides important supporting data for understanding the differences in fungal communities and physicochemical properties of medium-temperature Daqu across different regions.