The release of Phosphorus (P) from river sediments has been identified as a contributing factor to waters failing the criteria for ‘Good Ecological Status’ under the EU Water Framework Directive (WFD). To identify the contribution of sediment-P to river systems, an understanding of the factors that influence its distribution within the entire non-tidal system is required. Thus the aims of this work were to examine the (i) total (PTotal) and labile (PLabile) concentrations in sediment, (ii) the sequestration processes and (iii) the interactions between sediment P and the river water in the six non-tidal water bodies of the River Nene, U.K. Collection of sediments followed a long period of flooding and high stream flow. In each water body, five cores were extracted and homogenised for analysis with an additional core being taken and sampled by depth increments. Comparing the distribution of sediment particle size and PTotal data with soil catchment geochemical survey data, large increases in PTotal were identified in sediments from water body 4–6, where median concentrations of PTotal in the sediment (3603 mg kg−1) were up to double those of the catchment soils. A large proportion of this increase may be related to in-stream sorption of P, particularly from sewage treatment facilities where the catchment becomes more urbanised after water body 3. A linear correlation (r = 0.8) between soluble reactive phosphate (SRP) and Boron in the sampled river waters was found suggesting increased STW input in water bodies 4–6.PLabile concentrations in homogenised cores were up to 100 mg kg−1 PO4–P (generally < 2% of PTotal) and showed a general increase with distance from the headwaters. A general increase in Equilibrium Phosphate Concentrations (EPC0) from an average of 0.9–∼1.7 μm L−1 was found between water bodies 1–3 and 4–6. Fixation within oxalate extractable phases (Al, Fe and Mn) accounted for ∼90% of P binding in water bodies 4–6, but only between 31 and 74% in water bodies 1–3. Statistical models predicting PTotal (R2 = 0.78), oxalate extractable P (R2 = 0.78) and Olsen P (R2 = 0.73) concentrations in river sediments identified Mn oxy-hydroxides (MnOx) as a strong predictive variable along with the location within the river system. It is suggested that MnOx within model predictions is identifying a pool of mixed Fe–Mn oxy-hydroxides (MnOx–FeOOH) or Fe oxy-hydroxide (FeOOH) from the wider FeOxalate pool that are particularly effective at sorbing and fixing P. The findings demonstrate how sediment and P may accumulate along a 100 km non-tidal river system, the extent to which a range of processes can fix P within mineral phases and how natural flooding processes may flush sediment from the river channel. The processes identified in this study are likely to be applicable to similar river systems over their non-tidal water bodies in eastern England.