Phosphorus (P) reactivity and bioavailability in lake sediments may be determined by different forms of P and their distribution. Reactive and nonreactive P pools in two shallow subtropical lake sediments (Lake Apopka and Lake Okeechobee) were determined by sequential chemical extraction using 1 M NH4Cl (pH 7.0), 0.1 M NaOH, and 0.5 M HCl, reportedly representing loosely-bound P, Fe- and Al-bound P, and Ca- and Mg-bound P respectively. The sequential P fractionation was tested using pure P compounds and selected P minerals. The scheme effectively separated Fe- and Al-P from Ca-P fractions in an FePO4-AlPO4-Ca3(PO4)2 mixture. Readily available P, defined as the sum of water-soluble P and NH4Cl-extractable P, in the unconsolidated gyttja (UCG) layer (surface 0–30 cm) of Lake Apopka sediments accounted for 10.1 to 23.7% of total P (TP). This sediment P fraction constitutes a large reservoir which may act as a source of P to the overlying water. In subsurface marl layers (134–148 cm depth) of Lake Apopka, NH4Cl-P constituted <I% of TP whereas Ca-Mg-bound P and highly resistant P (residual P) accounted for 35 and 64% of TP respectively. Results suggest that 1 M NH4Cl (pH 7.0) and 0.5 M HCl, reported to dissolve carbonate-bound P and Ca-Mg-bound P, respectively, may not be extracting distinct pools of P. Lake Okeechobee mud sediments had low concentrations of readily available P (2% of TP) and were dominated by Ca-Mg-bound P (HCl-P≥58% of TP). Sediments in the littoral and peat areas of Lake Okeechobee, however, had high concentrations of readily available P (9.7 and 17.4% of TP respectively); hence, these sediments may play an important role in internal P cycling. The NaOH-P (Fe-Al-P) concentrations for Lake Okeechobee sediments were strongly correlated with amorphous and poorly-crystalline Fe (p< 0.01), suggesting that some P reactions in these sediments may be sensitive to changes in physico-chemical conditions such as redox potential and sediment resuspension.