The sources of the replenishment and hydrogeochemical evolution of acid mine drainage (AMD) from abandoned mines are issues of public concern around the world. To reveal the sources of groundwater replenishment and the nature of the hydrogeochemical processes that control the evolution of water quality in the multi-aquifer system of the abandoned Dashu pyrite mine in southwest China, the main control mechanisms of groundwater evolution are examined, based on hydrogeochemical analysis methods in combination with environmental isotope tracing methods, which in turn clarify the hydrogeochemical causes of groundwater pollution. According to the hydrogeochemical and stable and unstable isotope analyses, the diversity of groundwater hydrochemical types in the study area reflects the complexity of the groundwater hydrogeochemical environment, where groundwater is formed after the mixing of atmospheric precipitation and groundwater over multiple periods. The analysis of 2H, 18O, and T is used to identify the main sources of hydraulic connection between aquifers, groundwater, and mine water. The results show that there are close hydraulic connections between aquifers. Mine water and groundwater mainly come from the groundwater in the Quaternary accumulation platform. The results of the ion analysis and sulfur isotope tracing show that the main ions in the groundwater are derived from mineral dissolution/precipitation, cation exchange, pyrite oxidation, and other water-rock interaction processes. The sulfur in the groundwater mainly comes from the dissolution of gypsum, while the main source of sulfur in the mine water is the oxidation of pyrite, indicating that pyrite oxidation and cation exchange are the dominant processes in the mine water. The key hydrogeochemical processes were simulated using the reverse hydrogeochemical simulation method. The results show that the mining activities changed the water levels and flow conditions, strengthened the interaction between groundwater and aquifer lithology, which in turn affected the accompanying hydrogeochemical processes. After all of the mine was abandoned, it saw the cross-contamination between the aquifer and mine water. These results provide theoretical guidance for the identification of sources and key hydrogeochemical processes affecting groundwater and pollutants in the abandoned Dashu pyrite mines and similar abandoned mines with multiple aquifers, and can, therefore, provide technical support for the preparation of source prevention and control plans.