In the Riverina, NSW, Australia, abundant poultry litter (PL) could offer an alternative P nutrition source for irrigated cotton (Gossypium hirsutum L.). We assessed soil P availability, sequentially extracted P fractions, crop P uptake, and cotton yields using PL applications compared with mono‐ammonium phosphate (MAP) fertilizer in an alkaline Vertosol during the 2020–2021 season. Treatments were control, 150 kg MAP ha–1, 150 kg MAP ha–1 plus 4 Mg PL ha–1, and 4, 10, and 15 Mg PL ha–1. Crop P uptake (22–38 kg ha–1) at defoliation, seed P export (14–20 kg ha–1), P use efficiency (5.1–6.6 kg seed‐P Mg–1 lint), and lint yield (2,654–3,118 kg ha–1) were not different across treatments. Apparent recovery of P treatments was low (<15%). Across treatments, soil Colwell‐P (0.5 M NaHCO3) were invariably high (23–70 mg kg–1) during crop‐growing season and did not differ from planting to first‐flower. Residual inorganic‐P (conc‐HCl‐Pi + H2SO4‐H2O2‐Pi) declined by 12% with concurrent increases in labile NaHCO3‐Pi (4%) and organic‐P (dil‐HCl‐Po [4%] and conc‐HCl‐Po [3%]) over 18‐mo (from pre‐trial baseline sampling to defoliation) across treatments. At defoliation, the 15 Mg PL ha–1 treatment had lower soil pH and exchangeable sodium percentage and higher soil total C, moderately‐labile soil organic‐P (dil‐HCl‐Po), Colwell‐P, and extractable K than the control, MAP, or 4 Mg PL ha–1 treatments. Although P applications may not improve lint productivity because of inherent soil P release within a single cropping‐season, PL (15 Mg ha–1) may fully substitute for commercial MAP fertilizer for soil P maintenance and improving soil conditions in surface‐irrigated high‐yielding cotton in southeastern Australia.