Microbial communities were analyzed in six full-scale anaerobic digesters and a lab-scale digester using the random cloning method. The purposes were to confirm the applicability, reproducibility, and error range of this method; to discuss the difference in the dominant microbes determined by this method in different operational conditions of temperature (mesophilic and thermophilic) and substrate (garbage, sewage sludge, and livestock waste); and to determine key microbes in each digester. Each sample was analyzed in triplicate. In one of the samples, 373 clones were analyzed to study the composition of microbial community in the digester. Time course analysis was conducted from the start-up period for approximately one year in one of the digesters. Similar microbial diversity was obtained corresponding to the type of substrate change (sewage sludge to garbage). Operational taxonomic units (OTUs) closely related to Coprothermobacter sp. and unidentified bacterium clones TUG14 and TUG22 disappeared during the first 40 d, while OTUs closely related to Bacillus sp. and Clostridium sp. increased later. Microbial diversity in digesters is strongly affected by the operational conditions, and similar microbial diversity can be obtained in triplicate analysis and under similar operational conditions. The present study verified the applicability of this method to discuss the overall difference in microbial communities.