Water diversion rivers are the fundamental transport lines in water diversion projects. The changeable hydrological and physicochemical conditions lead to significant changes in biogeographical characteristics of microbial communities within river catchments. This study gives an insight into longitudinal patterns of the composition and diversity characteristics of microbial communities in water diversion rivers in the East Route of the South‐to‐North Water Diversion Project using 16S rRNA sequencing. A total of 937 743 microbial sequence reads are obtained and classified into 9178 distinct operational taxonomic units, belonging to 48 phyla and 424 genera, respectively. Along the water diversion line, obvious distribution characteristics of microbial communities could be observed in Actinobacteria, Firmicutes, Exiguobacterium, CL500‐29_marine_group, Acinetobacter, and Cloacibacterium. The relative abundance of both Actinobacteria and CL500‐29_marine_group increases from the upper rivers in the northern region to the lower rivers in the southern region, while Firmicutes and Exiguobacterium show the opposite trend. Higher microbial diversity is found in the upper rivers of the water diversion line than in the lower rivers. Externally, redundant analysis indicated the spatial shift in microbial community is largely shaped by ammonium, total nitrogen, conductivity, and total phosphorus. Internally, longitudinal construction of the microbial community is mainly affected by species replacement.