Diffuse phosphorus pollution is commonly remediated by diverting runoff through treatment wetlands to sequester phosphorus into soil layers. Much of the sequestered phosphorus occurs in organic forms, yet our understanding of its chemical nature is limited. We used NaOH-EDTA extraction and solution 31P NMR spectroscopy to speciate organic phosphorus sequestered in a large treatment wetland (STA-1W) in Florida, USA. The wetland was constructed on previously farmed peat and was designed to remove phosphorus from agricultural runoff prior to discharge into the Everglades. Unconsolidated benthic floc that had accumulated during the 9-year operation of the wetland was sampled along transects through two connected cells dominated by cattail (Typha dominigensis Pers.) and an additional cell colonized by submerged aquatic vegetation, including southern water nymph (Najas guadalupensis(Spreng.) Magnus) and coontail (Ceratophyllum demersum L.). Organic phosphorus was a greater proportion of the sequestered phosphorus in the cattail marsh compared to the submerged aquatic vegetation wetland, but occurred almost exclusively as phosphate diesters and their alkaline hydrolysis products. Itwas therefore markedly different from the organic phosphorus in mineral soils, which is dominated typically by inositol phosphates. Phosphate diesters are readily degradable in most soils, raising concern about the long-term fate of organic phosphorus in treatment wetlands. Further studies are now necessaryto assess the stability of the sequestered organic phosphorus in response to biogeochemical and hydrological perturbation.