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Antibiotics have attracted widespread attention around the world because they are ubiquitous in the environment and can lead to antibiotic-resistant microbes developing and pose ecotoxicological risks. In this study, we determined the spatiotemporal distributions of 39 antibiotics in 19 drinking water sources in Jiangsu area of the lower Yangtze River and attempted to identify the sources of the antibiotics and to prioritize the antibiotics. The total antibiotic concentrations in spring and fall were 234.56–6515.99 and 151.12–2562.59 ng/L, respectively. In spring, the total antibiotic concentration gradually increased from upstream to downstream. In fall, the antibiotic concentration did not markedly vary upstream to downstream (total concentrations 151.12–432.17 ng/L) excluding site S9 and S10. Analysis using a positive matrix factorization (PMF) model indicated that the antibiotics had four main sources. Pharmaceutical wastewater was the main source, contributing 34.1% and 41.2% of total antibiotics in spring and fall, respectively, and domestic wastewater was the second most important source, contributing 24.4% and 43% of total antibiotics in spring and fall, respectively. Pharmaceutical wastewater was the main source from midstream to downstream, but the other sources made different contributions in different areas because of the various ranges of human activities. An ecological risk assessment was performed. Stronger risks were posed by antibiotics in spring than fall, and fluoroquinolone antibiotics posed the strongest risks. Optimized risk quotients indicated that norfloxacin was a high-risk contaminant. An assessment of the risk of resistance development indicated that norfloxacin, ciprofloxacin, and enrofloxacin posed moderate to high risks of resistance development and should be prioritized for risk management. The results of this study are important reference data for identifying key sources of antibiotics and developing strategies to manage antibiotic contamination in similar areas. [Display omitted] • 39 antibiotics in drinking water sources from lower Yangtze River were firstly studied. • Total concentration gradually increased from upstream to downstream in spring. • The four potential sources of antibiotics were identified by the PMF model. • Pharmaceutical wastewater was critical sources, domestic wastewater was the second. • Priority antibiotics with high ecological and resistance risks were identified. [ABSTRACT FROM AUTHOR]