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Cryoconite holes, water-filled pockets containing biological and mineralogical deposits that form on glacier surfaces, play important roles in glacier mass balance, glacial geochemistry and carbon cycling. The presence of cryoconite material decreases surface albedo and accelerates glacier mass loss, a problem of particular importance in the rapidly melting Tibetan Plateau. No studies have addressed the microbial community composition of cryoconite holes and their associated ecosystem processes on Tibetan glaciers. To further enhance our understanding of these glacial ecosystems on the Tibetan Plateau and to examine their role in carbon cycling as the glaciers respond to climate change, we explored the bacterial communities within cryoconite holes associated with three climatically distinct Tibetan Plateau glaciers using Illumina sequencing of the V4 region of the 16S rRNA gene. Cryoconite bacterial communities were dominated by Cyanobacteria, Chloroflexi, Betaproteobacteria, Bacteroidetes and Actinobacteria. Cryoconite bacterial community composition varied according to their geographical locations, exhibiting significant differences among glaciers studied. Regional beta diversity was driven by the interaction between geographic distance and environmental variables; the latter contributed more than geographic distance to the variation in cryoconite microbial communities. Our study is the first to describe the regional-scale spatial variability and to identify the factors that drive regional variability of cryoconite bacterial communities on the Tibetan Plateau.