The mechanisms and rates of mercury methylation in the Florida Everglades are of great concern because of potential adverse impacts on human and wildlife health through mercury accumulation in aquatic food webs. We developed a new PCR primer set targeting hgcA, a gene encoding a corrinoid protein essential for Hg methylation across broad phylogenetic boundaries, and used this primer set to study the distribution of hgcA sequences in soils collected from three sites along a gradient in sulfate and nutrient concentrations in the northern Everglades. The sequences obtained were distributed in diverse phyla, including Proteobacteria, Chloroflexi, Firmicutes, and Methanomicrobia; however, hgcA clone libraries from all sites were dominated by sequences clustering within the order Syntrophobacterales of the Deltaproteobacteria (49 to 65% of total sequences). dsrB mRNA sequences, representing active sulfate-reducing prokaryotes at the time of sampling, obtained from these sites were also dominated by Syntrophobacterales (75 to 89%). Laboratory incubations with soils taken from the site low in sulfate concentrations also suggested that Hg methylation activities were primarily mediated by members of the order Syntrophobacterales, with some contribution by methanogens, Chloroflexi, iron-reducing Geobacter, and non-sulfate-reducing Firmicutes inhabiting the sites. This suggests that prokaryotes distributed within clades defined by syntrophs are the predominant group controlling methylation of Hg in low-sulfate areas of the Everglades. Any strategy for managing mercury methylation in the Everglades should consider that net mercury methylation is not limited to the action of sulfate reduction.