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Abstract Particle size greatly influences the fate of phosphorus (P) in estuaries as P adheres more readily to the larger surface area in smaller sized particles. Here, data on two size fractions of particulate matter, permanently suspended particulate matter (PSPM, ≤40 μm) and resuspended particulate matter (RSPM, >40 μm), from field and controlled laboratory erosion experiments were analyzed to determine their relative contribution to water column P in the mouth of the Susquehanna River in the upper Chesapeake Bay. Based on the composition of sequentially extracted P pools, C and N isotopes, and elemental data, all PSPM and the majority of RSPM are most likely derived from allochthonous sources through river transport. A minor fraction of particulate matter in the water column was derived from sediment resuspension, which had a dominant role above the sediment‐water interface in the river's mouth. The proportion of biologically available P pools to recalcitrant P pools in suspended particulate matter decreased with water column depth, indicating their preferential removal or biological utilization during settling. Suspended particulate matter (SPM) mobilized during sediment erosion experiments, regardless of particle size, was richer in biologically available P pools than SPM in the field, suggesting higher mobility of these pools in the field. These complementary results from field and field‐simulated laboratory erosion experiments provide unique insights into the composition of particulate matter under different hydrodynamic regimes in the river estuary.