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To our knowledge, ours is the first study to investigate the annual fluxes, environmental fate, and ecological risks of five categories of antibiotics from the Wuhan section of the Yangtze River (China). All the 24 antibiotics we tested for were detected in water, with total concentrations of 17.11–867.2 ng/L (mean: 63.69 ng/L), and 19 antibiotics were detected in sediment, at 0.02–287.7 ng/g (mean: 16.54 ng/g). Sulfonamides, amphenicols, and macrolides were the three most prominent antibiotic classes in water, and fluoroquinolones were the most prominent in sediment. Farming activities (animal husbandry and aquaculture) are proposed as the largest contributors to antibiotic pollution in the Wuhan section of the Yangtze River according to the Unmix model, followed by municipal wastewater and mixed sources. Higher pollution levels were observed downstream (combined discharge of these sources). Monthly monitoring data (12 months) were used to estimate antibiotic annual fluxes, with 101.5 t (uncertainty: 5.6%) in the Wuhan section of the Yangtze River. Risk assessments showed that erythromycin, clarithromycin, and azithromycin posed medium and high ecological risks and were found in 9%–35% and 1.8%–3.7% of all water samples, respectively; enrofloxacin, clarithromycin, azithromycin, florfenicol, and thiamphenicol posed medium resistance risks in 1.9%–16.7% of waters in the Wuhan section of the Yangtze River. Our results have filled data gaps on antibiotic sources, annual fluxes, and resistance risk in the Wuhan section of the Yangtze River and demonstrated the importance of further management of antibiotic use in the studied areas. Environ Toxicol Chem 2023;42:605–619. © 2022 SETAC [ABSTRACT FROM AUTHOR]