The spread of antibiotic resistance genes in river systems is an emerging environmental issue due to their potential threat to aquatic ecosystems and public health. In this study, we used droplet digital polymerase chain reaction (ddPCR) to evaluate pollution with clinically relevant antibiotic resistance genes (ARGs) at 13 monitoring sites along the main stream of the Weihe River in China. Six clinically relevant ARGs and a class I integron-integrase (intI1) gene were analyzed using ddPCR, and the bacterial community was evaluated based on the bacterial 16S rRNA V3–V4 regions using MiSeq sequencing. The results indicated Proteobacteria, Actinobacteria, Cyanobacteria, and Bacteroidetes as the dominant phyla in the water samples from the Weihe River. Higher abundances of bla_TEM, strB, aadA, and intI1 genes (10³ to 10⁵ copies/mL) were detected in the surface water samples compared with the relatively low abundances of strA, mecA, and vanA genes (0–1.94 copies/mL). Eight bacterial genera were identified as possible hosts of the intI1 gene and three ARGs (strA, strB, and aadA) based on network analysis. The results suggested that the bacterial community structure and horizontal gene transfer were associated with the variations in ARGs.