Rivers are a significant reservoir of antibiotic resistance genes (ARGs), yet the biogeographic pattern of riverine ARGs and its underlying driving forces remain poorly understood. Here, we used metagenomic approach to investigate the spatio-temporal variation of ARGs in two adjacent sub-watersheds viz. North River (NR) and West River (WR), China. The results demonstrated that Bacitracin (22.8 % of the total ARGs), multidrug (20.7 %), sulfonamide (15.2 %) and tetracycline (10.9 %) were the dominant ARG types. SourceTracker analysis indicated that sewage treatment plants as the main source of ARGs, while animal feces mainly contributed in spreading the ARGs in the upstream of NR. Random forest and network analyses confirmed that NR was under the influence of fecal pollution. PCoA analysis demonstrated that the composition of ARGs changed along with the anthropogenic gradients, while the Raup-Crick null model showed that homogenizing selection mediated by class 1 integron intI1 resulted in stable ARG communities at whole watershed scale. Structural equation models revealed that microbial community, grassland and several non-antibiotic micropollutants may also play certain roles in influencing the distribution of ARGs. Overall, the observed deterministic formation of ARGs in riverine systems calls effective management strategies to mitigate the risks of antibiotic resistance on public health.