In wetland habitats, periphyton is a common component of open-water areas with species assemblage determined by local water quality. Extracellular polymeric substances (EPS) secreted by algae and bacteria give structure to periphyton, and differences in EPS chemistry affect the functional roles of these polymers. The Florida Everglades provide a unique opportunity to study compositional differences of EPS from distinctive algal assemblages that characterize areas of differing water chemistry. Water conservation area (WCA)-1 is a soft-water impoundment; periphyton was loosely associated with Utricularia stems and amorphous in structure, with a diverse desmid and diatom assemblage, and varying cyanobacterial abundance. Extracellular polymers were abundant and were loosely cell-associated sheaths and slime layers in addition to tightly cell-associated capsules. The EPS were complex heteropolysaccharides with significant saccharide residues of glucose, xylose, arabinose, and fucose. Carboxylic acids were also prominent, while ester sulfates and proteins were small components. Structured, cohesive cyanobacteria-dominated periphyton was observed in WCA-2A, a minerotrophic impoundment, and filaments were heavily encrusted with calcium carbonate and detrital matter. EPS were primarily cell-associated sheaths, and polymer residues were dominated by glucose, xylose, fucose, and galactose, with uronic acids also a significant component of the polymers. Principal components analysis revealed that periphyton community assemblage determined the monosaccharide composition of EPS, which ultimately determines a range of biogeochemical processes within the periphyton.