Résumé : The main objective of this study was to investigate the antibiotic resistance (AR) levels in wastewater(WW) and the impact on the receiving river. Samples were collected once per season over one year in theWWof a hospital, in the raw and treated WWof two wastewater treatment plants (WWTPs), as well asupstream and downstream from the release ofWWTPs effluents into the Zenne River (Belgium). Culturedependentmethods were used to quantify Escherichia coli and heterotrophic bacteria resistant toamoxicillin, sulfamethoxazole, nalidixic acid and tetracycline. Six antibiotic resistance genes (ARGs) werequantified in both particle-attached (PAB) and free-living (FLB) bacteria. Our results showed thatWWTPsefficiently removed antibiotic resistant bacteria (ARB) regardless of its AR profile. The ARGs levels werethe highest in the hospital WWand were significantly reduced in both WWTPs. However, ARB and ARGsabundances significantly increased into the Zenne River downstream from the WWTPs outfalls. Thevariation in the relative abundance of ARGs through WW treatment differed depending on the WWTP,fraction, and gene considered. The sul1 and sul2 genes in PAB fraction showed significantly higherrelative abundances in the effluent compared to the influent of both WWTPs. This study demonstratedthat WWTPs could be hotspots for AR spread with significant impacts on receiving freshwater ecosystems.This was the first comprehensive study investigating at the same time antibiotics occurrence, fecal bacteria indicators, heterotrophic bacterial communities, and ARGs (distinguishing PAB and FLB) to assess AR levels in WW and impacts on the receiving river.