Converting poultry litter (PL) into biochar by slow pyrolysis is a promising approach for recycling organic waste with enhanced phosphorus (P) utilization efficiency, which needs fundamental knowledge of in situ P speciation transformation for optimizing the biochar conversion conditions. In this study, solid-state NMR spectroscopy was employed to characterize solid-state P and C speciation of raw PL and PL-derived biochars prepared at various pyrolysis temperatures. The NMR analysis indicated that phytates were decomposed while hydroxyapatite (Ca10(PO4)6(OH)2) formed during conversion of PL to PL-derived biochar at a pyrolysis temperature above 300 °C. With increasing pyrolysis temperature to above 500 °C, farringtonite (Mg3(PO4)2) formed. The higher pyrolysis temperature also favored the formation of calcite and provided deeper carbonization (i.e., greater dominance of thermally stable aromatic structures) in the biochar products. Water extractable P decreased significantly from 2.9 g/kg in PL to less than 0.3 g/kg in the PL-derived biochars prepared above 300 °C, indicating the inhibition effect of pyrolysis on the P lability mainly through transformation of labile phosphates in PL into less soluble forms. Overall, this study suggested that different pyrolysis temperatures should be considered for selective conversion of PL to biochar products with distinct agricultural and environmental applications that demand special P release patterns.