Phosphorus (P) fractionation and distribution in sediments are of great concern in the Florida Everglades ecosystem because potential eutrophication of surface waters usually results from P external loading and stability. Intact core sediment samples were collected to a depth of 35 cm from wetlands and canals across Water Conservation Area 3 (WCA‐3) of the Florida Everglades. These sediment cores were sliced into 5 cm increments and analyzed for P contents in different fractions by sequential extraction. These fractions mainly included total P (TP), readily available P (Pi‐KCl), Fe/Al‐bound P (Pi‐NaOH), Ca/Mg‐bound P (Pi‐HCl), organic P (Po‐NaOH), and residual P (PoResidue). Results showed that the canal sediments had the highest concentrations of TP, with about 87% in the form of Ca/Mg‐bound fraction, and the concentrations of TP in these sediments increased with depth. In contrast, the wetland sediments contained the lowest concentrations of TP (predominantly in the organic fraction), with 43% residual P and 27% Po‐NaOH, and the concentrations of TP in these sediments decreased with depth. In addition, a large amount of the readily available P (up to 1500 mg kg−1) in the canal sediments was accumulated at the top layer of 0–5 cm. This study suggests that any disturbance and/or environmental alterations, such as high canal flow and dredging in canal sediments, could pose a potential risk of a P increase in the water column and, consequently, in the wetlands because of the release of readily available P despite the relatively stable nature of such P fractions in these sediments.