Constructed wetlands are used to treat acid drainage from surface or underground coal mines. However, little is known about the microbial communities in wetlands that receive Acid Mine Drainage (AMD). The purpose of this study was to characterize microbial communities in a constructed AMD wetland using molecular methods of microbial ecology. Samples of Fe(III)-precipitates were collected from the oxic sediment zone of a constructed wetland cell in southeastern Ohio that was treating acid drainage from an underground coal mine seep. The pH of the samples ranged between 2.1 and 3.9. Samples were pretreated with ammonium oxalate to remove interfering iron, and the DNA was extracted and purified by agarose gel electrophoresis prior to amplification of portions of the 16S rRNA gene. Amplified products were characterized by (1) Denaturing Gradient Gel Electrophoresis (DGGE) and (2) Restriction Fragment Length Polymorphism (RFLP), and the results of the microbial community composition were confirmed by Fluorescence In-Situ Hybridization (FISH) analysis. Viable counts of acidophilic iron and sulfur oxidizers and heterotrophs were also determined by a Most-Probable-Number (MPN) method. DNA from seven distinct DGGE bands was excised from the gel and sequenced. The sequences were matched to sequences in the GenBank bacterial 16S rDNA database. The DNA in two of the bands yielded matches with Acidithiobacillus ferrooxidans and the DNA in each of the remaining five bands was consistent with one of the following bacteria: Acidithiobacillus thiooxidans, strain TRA3-20 (a eubacterium), strain BEN-4 (an arsenite-oxidizing bacterium), an Alcaligenes sp., and a Bordetella sp. Fifteen bacterial species were identified by RFLP followed by 16S rRNA gene sequencing of which two were novel (<95% match). The closest matches to known bacterial species were autotrophic iron- and sulfur-oxidizing bacteria (A. ferrooxidans and A. thiooxidans), heterotrophic iron-oxidizing bacteria (TRA2-10, TRA3-20, and TRA5-3) and heterotrophic Stenotrophomas maltophilia, Bordetella spp., Alcaligenes sp., Alcaligenes faecalis, and Alcaligenes xylososidans. A. ferrooxidans (40% of total 16S rRNA gene clone library) and A. thiooxidans (35%) were identified as the dominant bacterial species in wetland precipitates. The FISH analysis revealed that the most numerous bacterial species in this wetland system was A. ferrooxidans, comprising up to 37% of the bacterial population. A. thiooxidans was also abundant. Heterotrophs in the Acidiphilium genus totaled 20% of the bacterial population. Leptospirillum ferrooxidans was not detected in the bacterial community. The low bacterial diversity in these samples reflects the highly inorganic nature of the oxic sediment layer where high numbers of iron- and sulfur-oxidizing bacteria would be expected. A continuous, abundant supply of reduced iron and sulfur compounds, but with limited organics originating from the coal seam or washed off from the vegetation and soil surrounding the inlet channel to the wetland, control the microbial community. Both molecular and culture methods revealed the dominant bacteria in this acid receiving, oxic wetland were A. thiooxidans and A. ferrooxidans. However, MPN counts using culture methods were only a fraction of the corresponding FISH counts, suggesting many A. thiooxidans and A. ferrooxidans species, while closely related genetically, remain unculturable.