Tailings management in the oil sands industry aims to increase the rate of densification of fine clay and silt particles, releasing pore water for reuse in bitumen extraction and producing mature fine tailings (MFT) for subsequent use in reclamation. Microbial methane production has accelerated densification of the MFT, and acetate amendment may enhance this process. To identify important microorganisms in methane production, 16S rRNA gene sequences were used. At 22°C, methanogenic cultures from acetate-amended or unamended tailings comprised hydrogenotrophic methanogens and clostridial homoacetogens, regardless of acetate amendment. These groups could be participating in syntrophic acetate oxidation. In MFT samples collected from various depths of two active tailings ponds at the Syncrude Canada Mildred Lake site most methanogens were related to acetoclastic Methanosaeta spp. Most Bacteria were related to the Proteobacteria, including sulfate-reducing bacteria and hydrocarbon degraders. Based on these results, a possible pathway for methane production from MFT is proposed.