Lakes offer a primary setting for the collection and spread of antibiotic resistance genes (ARGs) within natural habitats, so it is of great importance and urgency to investigate the characteristics of ARGs in such aquatic ecosystems. Herein, the occurrence and distribution of microbial populations, ARGs, and mobile genetic elements (MGEs) in Poyang Lake, the largest freshwater lake in China, were analyzed in-depth through metagenomic sequencing. Surface water and sediment samples were examined simultaneously at 10 sampling sites covering the inlets of five tributaries and central lake areas. Results showed that the antibiotic concentration was at a low level in both surface water and sediment. Proteobacteria and Actinobacteria were the two most prevalent phyla across all sampling sites. The surface water displayed a greater relative abundance of ARGs than the sediment. Multidrug resistance genes were the most abundant and diverse ARGs. Gene uppP was the most abundant ARG in both the surface water and sediment samples. ARGs in the sediment showed a decreasing trend from inlet to outlet of Poyang Lake, while in the water, the relative abundance of ARGs increased first and then decreased from inlet to outlet. ARG levels in Poyang Lake are thought to be closely related to human activity. A broad spectrum of MGEs series was recognized in both surface water and sediment samples; the abundance of MGEs was consistent with ARGs’ abundance. Additionally, the makeup of the microbial community significantly influenced the resistome, suggesting that the composition of ARGs was largely affected by the microbial community. These findings indicate that lake water can act as a significant medium for ARGs dispersion, and the clustering and spread of ARGs are prone to the effects of human activities in aquatic habitats.