Abstract: The overall water resources in ecologically fragile mining areas in our country are scarce, but in some areas, mine water inflow is exceptionally large. The identification of abnormal mine water sources in ecologically fragile mining areas is of great significance for the protection of the ecological environment in the mining areas. This study takes the Yushuwan coal mine with a water inflow exceeding 1000 m³/h as the research background. Based on the analysis of the characteristics of mine water inflow, 30 water samples were collected from the main aquifers and mine water inflow points in the study area, and high-throughput microbial sequencing of water samples was conducted. The sequencing results were analyzed using Alpha diversity analysis, Beta diversity analysis, and differential analysis of microbial composition to systematically study the characteristics of microbial communities in mine water bodies and analyze the sources of mine water filling. The research results indicate that the high-throughput test results have passed the Coverage index test, verifying its feasibility for identifying mine water filling. However, water samples that have close contact with human activities show a greater abundance of microorganisms, which need to be effectively avoided in sampling. The abundance and diversity of microorganisms in the shallow loose aquifer of Yushuwan Coal Mine are relatively high, with the highest abundance being found in genera such asNovosphingobium,Fusobacterium, andSulfuricum. The microbial richness of the bedrock aquifer represented by the Zhiluo Formation is relatively low, but the microbial diversity is high, with the highest microbial abundance being found in genera such asComamonas,Vermiphilaceae, andPaeniglutamicacter. The most dominant phylum among all water samples isProteobacteria, accounting for 35.5% to 89.7%. Alpha diversity, PCoA, and NSDM analysis revealed that the water inflow in the Yushu Bay mine is mainly composed of loose layers and Zhiluo Formation. However, Beta diversity analysis found that with the release of static reserves in the Zhiluo Formation, the water source for filling the loose layer of Lishi loess further increases. Comprehensive analysis shows that the Yushuwan coal mine currently adopts a water filling mode of rapid release of bedrock aquifer and continuous release of Lishi Formation aquifer. The statistical analysis results of mine water inflow confirm this conclusion. The statistical analysis results of mine water inflow confirm this conclusion. This study provides a new method for identifying water filling in aquifers with similar chemical compositions.