We examined concentrations and sulfur isotopic ratios (³⁴S/³²S, expressed as δ³⁴S in parts per thousand [‰] units) of sulfate in surface water, ground water, and rain water from sites throughout the northern Everglades to establish the sources of sulfur to the ecosystem. The geochemistry of sulfur is of particular interest in the Everglades because of its link, through processes mediated by sulfate-reducing bacteria, to the production of toxic methylmercury in this wetland ecosystem. Methylmercury, a neurotoxin that is bioaccumulated, has been found in high concentrations in freshwater fish from the Everglades, and poses a potential threat to fish-eating wildlife and to human health through fish consumption. Results show that surface water in large portions of the Everglades is heavily contaminated with sulfate, with the highest concentrations observed in canals and marsh areas receiving canal discharge. Spatial patterns in the range of concentrations and δ³⁴S values of sulfate in surface water indicate that the major source of sulfate in sulfur-contaminated marshes is water from canals draining the Everglades Agricultural Area. Shallow ground water underlying the Everglades and rain water samples had much lower sulfate concentrations and δ³⁴S values distinct from those found in surface water. The δ³⁴S results implicate agricultural fertilizer as a major contributor to the sulfate contaminating the Everglades, but ground water under the Everglades Agricultural Area (EAA) may also be a contributing source. The contamination of the northern Everglades with sulfate from canal discharge may be a key factor in controlling the distribution and extent of methylmercury production in the Everglades.