The vinasse is a residue of ethanol production with the potential for methane production, requiring an allochthonous inoculum. Several microorganisms act in the different phases of anaerobic digestion, and the identification of these microbial communities is essential to optimize the process. The characterization of the microbiota involved in the biodigestion of vinasse was observed in the initial stage (IS), at the peak of methane production (MS) and the end of the process (FS) of the best performance assay by high-throughput sequencing. The highest methane production was 0.78 mmolCH4.gVS.h-1 at 243.7 h in the substrate/inoculum ratio of 1.7, with consumption partial of acetic, propionic and isobutyric acids and an 82% reduction of chemical oxygen demand. High microbial diversity was found. The genera Clostridium, Acinetobacter, Candidatus Cloacamonas, Bacteroides, Syntrophomonas, Kosmotoga, the family Porphyromonadaceae and the class Bacteroidia were the most abundant in the maximum methane production. Methane production was driven by Methanobacterium and Methanosaeta, suggesting the metabolic pathways used were hydrogenotrophic and acetoclastic.