Huangbai River, including four enchained reservoirs, is located in a phosphate-rock watershed in Yichang City, Central China. Previous studies showed that pollution levels due to activities of phosphate mining (PM) were extremely high and the water quality of reservoirs was severely affected by sedimentation. Yet, detailed knowledge of the sediment is lacking at that high phosphorus (P) concentration basin. Therefore, the different P fractions and its diffusion fluxes at the water-sediment interface of four deep reservoirs have been investigated for the first time to understand the internal P loading. The results indicated that the concentrations of total P , ranging from 9,631.5 to 581.6 mg kg−1, exhibited an apparent spatial trend from the upstream to the downstream reservoirs, which corresponded with the activities of P mining in this basin. The P-fraction concentrations obtained from the sites were mainly ranked in this order: P associated with calcium (Ca-Pi) > organic P (Po) > P bound to aluminum (Al), ferrum (Fe) and manganese (Mn) oxides and hydroxides (Fe/Al-Pi). The orthophosphate diffusion fluxes ranged from −0.40 ± 0.09 to 0.95 ± 0.14 mg m−2 d−1 which were positively related to P fraction concentrations while negatively related to oxidation–reduction potential and dissolved oxygen values. These results implied the dynamics role of internal P loading. The principle component analysis suggested that PM activities and internal P loading were the most reactive factors in this river system.