Fujian reservoirs in southeast China are important water resources for economic and social sustainable development, although few have been studied previously. Dongzhen Reservoir, a typical subtropical stratified reservoir in Fujian, was chosen for investigation of bacterial distribution, composition and diversity in autumn 2011. As the only built large reservoir in Putian City, it plays an important role in irrigation, flood control, hydroelectric power, and water supply. Dongzhen Reservoir shows an obvious phenomenon of water thermal stratification during summer and autumn, thus creates a unique gradient of environmental variables along the water column. We investigated the bacterial community and its relation to environmental variables in this study for a better understanding of vertical distribution of bacterial community and the primary environmental drivers in a stratified reservoir. Five water samples were collected from five different depths according to the vertical changes of temperature. Both PCR-DGGE and sequencing were used to investigate the bacterial community and diversity. Moreover, physical and chemical parameters were measured according to the national standard methods. Multivariate statistical techniques were used to examine the relationship between bacterial community and environmental variables. Principal component analysis (PCA) clearly showed that water temperature, dissolved oxygen (DO), chlorophyll a (Chl a), total nitrogen (TN), ammonia nitrogen (NH₄-N), nitrite/nitrate nitrogen (NO_x-N) and electric conductivity of the upper aerobic zone were differed from those of the deeper anoxic zone. Both the number of DGGE bands and the Shannon-Wiener index of the deeper anoxic zone were higher than those of the upper aerobic zone. Further, two groups were distinguished by the cluster analysis of bacterial communities based on the Bray-Curtis similarity. Thus, Dongzhen Reservoir presented an obviously physical, chemical, and biological stratified phenomenon. Seven bands that common to all sampling depths were extracted and sequenced, and among which four were identified as Betaproteobacteria, indicating that Betaproteobacteria were the most dominant taxa in Dongzhen Reservoir in autumn. Bacterial community composition and diversity differed greatly among different sampling depths, and these differences were closely related to the physical and chemical stratification of the water body. Redundancy analysis (RDA) demonstrated DO was the significant environmental variable that shaping the bacterial community and diversity (P < 0.01). Therefore, we should pay more attention to DO and thermal stratification of reservoirs for sustainable reservoir management.