We monitored bacterioplankton communities from Chesapeake Bay over 2 years (2002–2004) by use of denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene. Chesapeake Bay bacterioplankton exhibited a repeatable annual pattern and strong seasonal shifts. In winter, the bacterial communities were dominated by Alphaproteobacteria and Actinobacteria, whereas in summer, the predominant bacteria were members of Alphaproteobacteria, Gammaproteobacteria, Cyanobacteria, Actinobacteria, Planctomycetes, and Bacteroidetes. Phylotypes of Alphaproteobacteria and Actinobacteria present in warm seasons were different from those in cold seasons. Relatively stable communities were present in summer–fall across the sampling years, whereas winter communities were highly variable interannually. Temporal variations in bacterial communities were best explained by changes of chlorophyll a (Chl a) and water temperature, but dissolved oxygen, ammonia, nitrite and nitrate, and viral abundance also contributed significantly to the bacterial seasonal variations.