The sub-lakes of Poyang Lake have significant ecological value. To date, the diazotrophs in sub-lakes are unknown. Moreover, no study has simultaneously focused on diazotrophic communities in the water and sediment in natural freshwater ecosystems. In this study, we investigated the diazotrophic alpha diversity, structure, abundance, molecular ecological network, and community assembly mechanism in the water and sediment of sub-lakes using Illumina MiSeq sequencing and a quantitative polymerase chain reaction assay. The results showed that the sediment had a greater diversity of diazotrophs than the water. Proteobacteria and Spirochaetes were the dominant diazotrophic phyla in the water, whereas Proteobacteria was the dominant diazotrophic phylum in the sediment. There were significant differences in the composition of diazotrophic communities between the water and sediment. The sediment had a more complex co-occurrence network of diazotrophs than the water. Deterministic processes dominate the community assembly of diazotrophs in both the water and sediment of the sub-lakes, and the relative role of deterministic processes was stronger for sediment than water. Our study is the first to reveal the differences in the diazotrophic communities between the water and sediment in natural freshwater ecosystems and provides the fundamental scientific datasets for understanding the nitrogen fixation process in sub-lakes.