Phosphorus (P) fractionation is critical to understand P cycling in sediment and across the sediment–water interface. To examine the effect of pretreatment on the P fractionation of anaerobic sediment, anaerobic sediment samples were treated by oven-drying, air-drying, and freeze-drying, and a fresh sediment sample was used as a control treatment. A five-step sequential extraction procedure was applied, and six P fractions were examined. The results indicated that PNaCl (P extracted by NaCl), PBD (P extracted by Na2S2O4 and NaHCO3), PNaOH (P extracted by NaOH), and organic P were significantly influenced by pretreatment, whereas PHCl (P extracted by HCl) and residual P were not significantly affected by pretreatment. Compared to fresh sediment, drying treatments decreased PNaCl, PNaOH, and organic P but increased PBD. Among the three drying methods, oven-drying resulted in the greatest change in P fractionation; the effects of air-drying and freeze-drying were similar to each other. The total extracted P were similar among the four pretreatment methods. For the measurement of accurate P fractions, fresh sediment is the best option.