Microorganisms in acid mine drainage are typically acidophiles that mediate the oxidation of reduced compounds of iron and sulfur. However, microbial populations in wetland systems constructed to treat acid mine drainage are not well characterized. The purpose of this study was to analyze bacterial diversity, using cultivation-independent molecular ecological techniques, in a constructed wetland that received acid drainage from an abandoned underground coal mine. DNA was purified from Fe(III)-precipitates from the oxidized surface zone of wetland sediments and 16S rRNA gene sequences were amplified and cloned. A total of 200 clones were analyzed by restriction fragment length polymorphism (RFLP) and 77 unique RFLP patterns were obtained with four restriction enzymes. Of these patterns, 30 most dominant unique clones were selected for sequencing of their 16S rRNA genes. Half of these 30 clones could be matched with autotrophic iron- and sulfur-oxidizing bacteria (Acidithiohacillus ferrooxidans and Acidithiobacillus thiooxidans). Several clones also formed a clade with heterotrophic iron-oxidizing bacteria (TRA2-10, TRA3-20, and TRA5-3) and heterotrophic bacteria (Stenotrophomas maltophilia, Bordetella spp., Alcalgenes sp., Alcaligenesfaecalis, and Alcaligenes xylosoxidans). Approximately 40% and 35% of the analyzed RFLP restriction patterns were consistent with A. ferrooxidans and A. thiooxidans, respectively. The relatively high frequency of acidithiobacilli is consistent with the chemical and physical characteristics of this site-i.e., continuous, abundant supply of reduced iron and sulfur compounds, pH 3-4, ambient temperature, and limited organics originating from the coal seam and from vegetation or soil surrounding the inlet channel to the wetland. The RFLP results were consistent with our previous culture-independent PCR-DGGE and FISH study, showing relatively low bacterial diversity and predominance of mesophilic acidithiobacilli in oxic wetland sediments.