Neutrophilic iron-oxidizing bacteria (FeOB) are important catalysts of iron cycling in wetland environments. However, little is known about their diversity and distribution in various environments. The aim of this study was to develop a PCR-DGGE assay enabling the detection of neutrophilic iron oxidizers in wetland habitats. Gradient tubes were used to enrich FeOB. From these enrichments, a clone library was established on the basis of the almost complete 16S rRNA gene using the universal bacterial primers 27f and 1492r. This clone library consisted of mainly - and -Proteobacteria, among which two major clusters were closely related to Gallionella spp. Specific probes and primers were developed on the basis of this 16S rRNA gene clone library. The newly designed Gallionella-specific 16S rRNA gene primer set 122f/998r was applied to community DNA obtained from three contrasting wetland environments, followed by Denaturing Gradient Gel Electrophoresis (DGGE) analysis. A second 16S rRNA gene clone library was constructed using the PCR products from one of our sampling sites amplified with the newly developed primer set 122f/998r. The cloned 16S rRNA gene sequences all represented novel culturable iron oxidizers most closely related to Gallionella sp. On the basis of their nucleotide sequences, four groups could be identified that were comparable to the DGGE banding pattern obtained before with the same PCR products as used for the second clone library. Using these Gallionella-specific 16S rRNA gene-based primers, in combination with DGGE, first insights into the diversity and distribution of these bacteria in wetland soils were obtained.