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The effects of microwave (MW) pretreatment were investigated by six anaerobic digesters operated under thermophilic and mesophilic conditions at high organic loading rates (4.9–5.7 g volatile solids/L/d). The experiments and analyses were mainly designed to reveal the impact of MW pretreatment and digester temperatures on the process stability and microbial community structure by correlating the composition of microbial populations with volatile fatty acid (VFA) concentrations. A slight shift from biogas production (with a reasonable methane content) to VFA accumulation was observed in the thermophilic digesters, especially in the MW-irradiated reactors. Microbial population structure was assessed using a high-throughput sequencing of 16S rRNA gene on the MiSeq platform. Microbial community structure was slightly affected by different MW pretreatment conditions, while substantially affected by the digester temperature. The phylum Bacteroidetes proliferated in the MW-irradiated mesophilic digesters by resisting high-temperature MW (at 160 °C). Hydrogenotrophic methanogenesis (mostly the genus of Methanothermobacter) was found to be a key route of methane production in the thermophilic digesters, whereas aceticlastic methanogenesis (mostly the genus of Methanosaeta) was the main pathway in the mesophilic digesters.