e Located in southwest China, the Aha watershed is continually contaminated by acid mine drainage (AMD) produced from upstream abandoned coal mines. The watershed is fed by creeks with elevated concentrations of aqueous Fe (total Fe > 1 g/liter) and SO 4 2؊ (>6 g/liter). AMD contamination gradually decreases throughout downstream rivers and reservoirs, creating an AMD pollution gradient which has led to a suite of biogeochemical processes along the watershed. In this study, sediment samples were collected along the AMD pollution sites for geochemical and microbial community analyses. High-throughput sequencing found various bacteria associated with microbial Fe and S cycling within the watershed and AMD-impacted creek. A large proportion of Fe-and S-metabolizing bacteria were detected in this watershed. The dominant Fe-and S-metabolizing bacteria were identified as microorganisms belonging to the genera Metallibacterium, Aciditerrimonas, Halomonas, Shewanella, Ferrovum, Alicyclobacillus, and Syntrophobacter. Among them, Halomonas, Aciditerrimonas, Metallibacterium, and Shewanella have previously only rarely been detected in AMD-contaminated environments. In addition, the microbial community structures changed along the watershed with different magnitudes of AMD pollution. Moreover, the canonical correspondence analysis suggested that temperature, pH, total Fe, sulfate, and redox potentials (E h ) were significant factors that structured the microbial community compositions along the Aha watershed.A cid mine drainage (AMD) is one of the most environmentally threatening by-products of the mining industry. The chemical and/or microbial weathering of metal sulfide-rich rocks such as pyrite (FeS 2 ), sphalerite (ZnS), and galena (PbS) produces sulfuric acid, which leads to the formation of AMD. AMD typically has low pH and elevated concentrations of sulfate and metals, which severely impair water and soil quality (1, 2).Despite the extreme toxicity and acidity, the AMD-associated environments harbor numerous acidophilic microorganisms (3, 4). Recently, molecular techniques have been extensively applied to study AMD microbiology, and a variety of acidophilic and metal-tolerant microorganisms have been identified (5). Prokaryotic microorganisms that are metabolically active in acidic niche boundaries (pH Ͻ 3) were found to be phylogenetically diversified, affiliated within the phyla Proteobacteria, Firmicutes, Acitinobacteria, Nitrospirae, and Acidobacteria (5). Many of these acidophiles are capable of Fe and S cycling (e.g., oxidation and reduction of these elements in appropriate geochemical and microbial environments) and thus play an important role both in the formation of acid mine waters and in natural attenuation of AMD. However, our understanding of the roles of geochemical factors in shaping microbial community structure and the potential of microorganisms in natural attenuation of AMD and AMD-impacted watersheds is still limited.The AMD of the Aha watershed in southwest China provides an ideal opportunity to ...