Abstract Background This study aims to chemically characterize thin stillage derived from lignocellulosic biomass distillation residues in terms of organic strength, nutrient, and mineral content. The feasibility of performing anaerobic digestion on these stillages at mesophilic (40 °C) and thermophilic (55 °C) temperatures to produce methane was demonstrated. The microbial communities involved were further characterized. Results Energy and sugar cane stillage have a high chemical oxygen demand (COD of 43 and 30 g/L, respectively) and low pH (pH 4.3). Furthermore, the acetate concentration in sugar cane stillage was high (45 mM) but was not detected in energy cane stillage. There was also a high amount of lactate in both types of stillage (35–37 mM). The amount of sugars was 200 times higher in energy cane stillage compared to sugar cane stillage. Although there was a high concentration of sulfate (18 and 23 mM in sugar and energy cane stillage, respectively), both thin stillages were efficiently digested anaerobically with high COD removal under mesophilic and thermophilic temperature conditions and with an organic loading rate of 15–21 g COD/L/d. The methane production rate was 0.2 L/g COD, with a methane percentage of 60 and 64, and 92 and 94 % soluble COD removed, respectively, by the mesophilic and thermophilic reactors. Although both treatment processes were equally efficient, there were different microbial communities involved possibly arising from the differences in the composition of energy cane and sugar cane stillage. There was more acetic acid in sugar cane stillage which may have promoted the occurrence of aceticlastic methanogens to perform a direct conversion of acetate to methane in reactors treating sugar cane stillage. Conclusions Results showed that thin stillage contains easily degradable compounds suitable for anaerobic digestion and that hybrid reactors can efficiently convert thin stillage to methane under mesophilic and thermophilic conditions. Furthermore, we found that optimal conditions for biological treatment of thin stillage were similar for both mesophilic and thermophilic reactors. Bar-coded pyrosequencing of the 16S rRNA gene identified different microbial communities in mesophilic and thermophilic reactors and these differences in the microbial communities could be linked to the composition of the thin stillage.