The phosphorus (P) composition of sediment profiles in three subtropical lakes of contrasting trophic state in Florida, USA, was determined by sequential fractionation and solution 31 P NMR spectroscopy. Sediment from Lake Annie, an oligo-mesotrophic sinkhole with moderately acidic sediment (pH 5.4; loss on ignition 58 %), contained higher total P concentrations than sediment from eutrophic Lake Okeechobee (pH 7.7, loss on ignition 36 %) and hyper-eutrophic Lake Apopka (pH 7.5, loss on ignition 69 %). The chemical nature of sediment P varied markedly among the three lakes, suggesting the predominance of different diagenetic processes. Lake Okeechobee sediment was dominated by inorganic P, indicating the dominance of abiotic reactions; Lake Annie sediment contained abundant organic P throughout the sediment profile, indicating the importance of organic P stabilization at acidic pH; Lake Apopka contained almost half of its sediment P in microbial biomass, indicating the importance of biotic processes in regulating P dynamics. Solution 31 P NMR spectroscopy of NaOH-EDTA extracts revealed that organic P occurred mainly as phosphomonoesters in all lakes. However, sediment from Lake Apopka also contained abundant phosphodiesters and was the only lake to contain detectable concentrations of polyphosphate, perhaps due to a combination of alternating redox conditions and high concentrations of inorganic phosphate and organic carbon. Organic P concentrations determined by sequential fractionation and solution 31 P NMR spectroscopy were similar for all lakes when microbial P was included in values for sequential fractionation. We conclude that the chemical nature of sediment P varies markedly depending on trophic state and can provide important information on the dominant processes controlling P cycling in subtropical lakes.