Rivers are considered a vital reservoir of antibiotic resistance genes (ARGs) and are critical to disseminate ARGs. The present study delved into the ARGs pollution of the sediments in the upper reaches of Huaihe river, one of the seven longest rivers in China, by high-throughput quantitative PCR. Subsequently, the relationship between ARGs and the bacterial community/mobile genetic elements (MGEs) was determined. As revealed from the results, the overall ARGs ranged from 2.65×10⁻³ to 6.14×10^-2/16S copies, and the abundance of ARGs in the tributaries was significantly higher than that in the mainstreams (p<0.05). Moreover, the ARGs introduced by tributaries were capable of affecting the whole mainstream of Huaihe river. As suggested from the results of co-occurrence analysis and pRDA analysis, MGEs were reported as the major driver to disseminate ARGs in the upper reaches of Huaihe river basin. The larger the MGEs proportion, the higher the likelihood of ARGs transferring from antibiotic resistance bacteria to human pathogens in Huaihe river.