The different drainage basins of large rivers such as the Mississippi River represent interesting systems in which to study patterns in freshwater microbial biogeography. Spatial variability in bacterioplankton communities in six major rivers (the Upper Mississippi, Missouri, Illinois, Ohio, Tennessee, and Arkansas) of the Mississippi River Basin was characterized using Ion Torrent 16S rRNA amplicon sequencing. When all systems were combined, particle-associated ({ extgreater}3 micron) bacterial assemblages were different from free-living bacterioplankton in terms of overall community structure, partly because of differences in the proportional abundance of sequences affiliated with major bacterial lineages (Alphaproteobacteria, Cyanobacteria, Planctomycetes). Both particle-associated and free-living communities ordinated by river system, a pattern that was apparent even after rare sequences or those affiliated with Cyanobacteria were removed from the analyses. Ordination of samples by river system correlated with environmental characteristics of each river, such as nutrient status and turbidity. Communities in the Upper Mississippi and the Missouri, and the Ohio and the Tennessee, pairs of rivers that join each other, contained similar taxa in terms of presence/absence data, but differed in the proportional abundance of major lineages. The most common sequence types detected in particle-associated communities were picocyanobacteria in the Syn/Pro clade, while free-living communities also contained a high proportion of LD12 (SAR11/Pelagibacter)-like Alphaproteobacteria. This research shows that while different tributaries of large river systems such as the Mississippi River harbor distinct bacterioplankton communities, there is also microhabitat variation such as that between free-living and particle-associated assemblages.