Excess phosphorus (P) applied to agricultural fields accumulates in the soil profile and is susceptible to leaching, subsequently impacting adjacent and downstream aquatic ecosystems. Planting a series of crops in a field over a given period of time is considered conservation crop rotation, which may reduce nutrient losses by reducing P inputs (i.e., including crops with lower P requirements) and increasing P demand (i.e., by increasing biodiversity, vegetative land cover, and length of the growing season). Here, we calculated P balances (Inputs–Outputs) in agricultural fields with corn-soybean rotations (CS) and corn-soybean-wheat rotations (CSW) to determine P accumulation or depletion. We then compared P losses, as dissolved reactive P (DRP) and total P (TP), between the CS and CSW rotations. Results indicated annual P deficits in CS (median = −9 kg P/ha) and CSW (median = −5 kg P/ha) fields. However, DRP and TP losses persisted and were significantly greater from CSW (median = 0.5 kg DRP/ha and 2 kg TP/ha) compared to CS fields (median = 0.1 kg DRP/ha and 1 kg TP/ha), likely due to elevated soil test phosphorus (STP) concentrations in CSW fields. The fraction of applied P exported from fields as both DRP and TP was also greater in CSW (DRP = 2%; TP = 9%) compared to CS (DRP = 1%; TP = 3%), further confirming more available P susceptible to loss. Findings here highlight the need for further research between conventional and conservation crop rotation on fields with similar STP concentrations and emphasize the importance of P application in accordance with fertility recommendations.