Periphyton samples from Water Conservation Areas, Big Cypress National Preserve, and Everglades National Park in south Florida were analyzed for concentrations of total mercury, methylmercury, nitrogen, phosphorus, organic carbon, and inorganic carbon. Concentrations of total mercury in periphyton decrease slightly along a gradient from north‐to‐south. Both total mercury and methylmercury are positively correlated with organic carbon, nitrogen and phosphorus in periphyton. In horizontal sections of periphyton mats, total mercury concentrations tend to be largest at the tops and bottoms of the mats. Methylmercury concentrations tend to be the largest near the bottom of mats. These localized elevated concentrations of methylmercury suggest that there are “hot spots”; of methylmercury in periphyton.