Biological nitrogen fixation, which is mediated by nitrogen-fixing microorganisms (diazotrophs), is an important source of fixed nitrogen in aquatic ecosystems. Recent advances in molecular biology techniques are leading to progress in the elucidation of the diazotrophic community possessing the nifH nitrogenase gene in various ecosystems. So far, the majority of studies of diazotrophic communities are based on marine samples, with freshwater habitats remaining largely unexplored. The aims of this study are to characterize the spatiotemporal patterns of a diazotrophic microbial community in a subtropical stratified reservoir (Tingxi Reservoir, southeast China), and to examine the relationships between the diazotrophic community structure and environmental variables. In this study, samples from the water column (five layers) were collected in July and October 2012, and in January and April 2013. Nitrogen-fixing microorganisms were studied using quantitative real-time PCR and clone library techniques. In Tingxi Reservoir, the water column was well mixed in winter, whereas there was clear and stable stratification in spring, summer, and autumn. Our results indicated that there were distinct spatial and seasonal patterns of abundance, activity, composition, and diversity in the diazotrophic community that was linked to water stratification in the Tingxi Reservoir. In total, 106 OTUs belonging to seven groups (i.e., 46 Cyanobacteria, 21 α-Proteobacteria, 3 β-Proteobacteria, 9 γ-Proteobacteria, 1 Firmicutes, 18 unidentified nitrogen-fixing bacteria, and 8 unknown taxa) were observed, the most diverse and dominant group of which was cyanobacteria. Both α- and γ-proteobacteria were much more common than β-proteobacteria in the reservoir.The deeper waters harbored a high percentage of unidentified bacteria and unknown taxa. The number of sequences similar to cyanobacteria in the surface water was higher than in the bottom water in spring, summer, and autumn. The Cyanobacteria OTUs number contributed more than 50% of the total OTUs in both surface and bottom waters in winter. Firmicutes were only detected in the surface water sample in October. Interestingly, our cluster analysis indicated that surface water diazotrophic communities in spring, summer, and autumn (with a stable stratification) first formed a group, then clustered with the bottom communities. However, both surface and bottom diazotrophic communities exhibited a relatively high similarity in winter due to water mixing. The pH, dissolved oxygen, and chlorophyll a showed a significant negative relationship with the DNA copy number, whereas the NO_x-N showed a strong positive correlation with the DNA copy number. Water temperature and NH₄-N had a negative significant relationship with RNA/DNA ratio and RNA copy number, respectively. It appeared that nitrogen-fixing bacteria showed distinctly nonrandom spatial and seasonal distributions in the Tingxi Reservoir, and their communities were either complexly structured by the thermal stratification or adapted to different environmental niches. Therefore, a stratification-based management strategy should be considered when developing methods for protecting drinking water quality and for controlling the cyanobacterial blooms.