The gene pair hgcAB is essential for microbial mercury methylation. Therefore, improved techniques for capturing hgcAB presence and diversity are necessary for identifying both the major players in environmental methylmercury production as well as the evolutionary history of the gene pair. This study applies next generation sequencing methods for using the genes as biomarkers for identifying key producers of the toxin in aquatic ecosystems. High-throughput sequencing of hgcAB amplicons from environmental samples were validated using mock communities. This dataset includes freshwater sediment samples from East Fork Poplar Creek (Oak Ridge, TN, USA) and mock communities produced from culture spikes into salt marsh sediment (Rhode River, MD, USA).