Pollution of water bodies by phosphorus in runoff from soil amended with animal manures is one of the greatest threats to water quality in developed countries. The environmental fate of manure phosphorus is determined in part by its chemical composition, yet extraction procedures to assess this are poorly developed and provide no structural information. We used solution 31P NMR spectroscopy to quantify phosphorus compounds in sequential extracts of three contrasting manures (broiler litter, beef-cattle manure, swine manure). Using a procedure originally developed for soils, but commonly applied to manures, phosphorus was extracted sequentially with deionized water, 0.5 M NaHCO3, 0.1 M NaOH, and 0.5 M HCl. Water and NaHCO3 extracted readily soluble compounds, including phosphate, phospholipids, DNA, and simple phosphate monoesters, which are mobile in soil and biologically available. In contrast, NaOH and HCl extracted poorly soluble compounds, including phytic acid (myoinositol hexakisphosphate). The latter is immobile in soil and of limited biological availability. Based on these results, we developed a simplified two-step fractionation procedure involving extraction of readily soluble phosphorus in 0.5 M NaHCO3 followed by extraction of stable phosphorus in a solution containing 0.5 M NaOH and 50 mM EDTA. This revised procedure separates manure phosphorus into structurally defined fractions with environmental relevance and will facilitate research on this important aspect of environmental science.