Nitrogen-fixing microorganisms (diazotrophs) play important roles in aquatic biogeochemistry and ecosystem functioning. However, little is known about the spatiotemporal variation of diazotrophic microbial communities in deep subtropical reservoirs. In this study, denaturing gradient gel electrophoresis (DGGE), clone libraries, quantitative PCR, and quantitative reverse transcription (RT)-PCR were used together to examine the vertical and seasonal patterns of diazotrophic microbial communities based on nitrogenase (nifH) gene sequences in the Dongzhen Reservoir, China, across time (every 3 months for 1 year) and space (five different water depths). In general, the numbers of DGGE bands increased with water depth during the stratification seasons (spring, summer, and autumn), with the clone-library-based operational taxonomic unit (OTU) number and nifH gene diversity being highest in autumn (6 OTUs at depth 0 m; 15 OTUs at 33 m) and winter (12 OTUs at 0 m, 13 OTUs at 33 m) but decreasing drastically in spring (2 OTUs at 0 m, 3 OTUs at 33 m) and summer (3 OTUs at 0 m, 2 OTUs at 33 m). The nifH gene abundance was lowest in the water mixing season (winter average, 5.17 × 10(7) copies/L) but increased in the three other seasons (9.03 × 10(9) copies/L). Cyanobacteria (dominated by filamentous thermophilic cyanobacteria and Cylindrospermopsis raciborskii) were the most dominant diazotrophic group at all depths and seasons, while both alphaproteobacteria and gammaproteobacteria were co-dominant in the bottom waters in autumn and winter. The distinct seasonal and spatial patterns in diazotrophic communities were significantly related to total nitrogen (TN) and ammonium nitrogen (NH4-N) in the reservoir (P < 0.01). Further, TN showed a significant positive correlation with nifH RNA copy number (P < 0.05) and DGGE band number (P < 0.01), whereas the NH4-N was negatively correlated with nifH DNA copy number (P < 0.01) and positively with both RNA/DNA ratio (P < 0.01) and DGGE band number (P < 0.01). Our data indicated that water stratification, mixing, and nitrogen might drive the diazotrophic community structure and activity in complex ways, thereby influencing the aquatic nitrogen cycle. Therefore, adaptive reservoir management strategies should carefully consider the effects of water stratification for protecting drinking water quality and for controlling the potential for diazotrophic cyanobacteria blooms.