Quantification of phosphorus transport and fate is necessary for effective mitigation to water body eutrophication issues but challenging in a large river basin due to the spatial heterogeneity of landscape patterns, especially in data-limited regions. This study presents a new estimation framework, taking usage of multi-source data sets locally and globally, to generate high-resolution maps of phosphorus sources, retention and delivery for a large river basin of Asia, the Yangtze River basin. Results show that 65%–73% phosphorus delivered to the river network and the Yangtze estuary originate from 26% basin areas, which are dominated by sources including urban living, livestock cultivation and cropland farming. 57% (30%–73%) phosphorus from sources retains in the basin rather than delivers to the estuary. Most (about 70%) retention occurs in main tributaries, lakes and reservoirs, posing high freshwater eutrophication risk. Small wetlands are identified as key terrestrial retention landscapes for diffuse sources, which serve as promising buffers if their functions are enhanced with proper restoration and better management. The new framework and its application support detailed understanding of phosphorus source, retention and delivery in a large river basin and provide insights into the impact of landscape patterns that helps enhancing eutrophication mitigation strategies.