Illustrations of the methanogenic pathways indicated by carbon isotope (δ13C) analysis under mesophilic and thermophilic conditions. Display omitted •The Methanogenesis pathway was quantitatively determined by 2–13C isotope.•SAO-HM account for 41% and 50% of methane formation at 37 °C and 55 °C.•The thermophilic digester had 15% lower methane yield rates.•Stable processes were achieved at free ammonia of 31 and 145 mg/L, respectively. The impact of temperature on the anaerobic digestion of chicken manure was investigated by studying the process performance and pathway for continuously-fed digesters under mesophilic and thermophilic conditions. The mesophilic digester obtained a 15% higher methane yield compared with the thermophilic digester. Mesophilic and thermophilic digester had free ammonia of 31 and 145 mg/L, respectively. The stable carbon isotope analysis indicated that 41% and 50% of acetate was converted to methane through the syntrophic acetate oxidation and hydrogenotrophic methanogenesis (SAO-HM) pathway under mesophilic and thermophilic conditions, respectively. The genus Pseudomonas represented 10% and 16% under mesophilic and thermophilic conditions, respectively. A high abundance of the methanogens genus Methanoculleus (94% of total methanogens) in mesophilic and the genus Methanothermobacter (96%) in thermophilic digesters indicated they were the main hydrogenotrophic partners in SAO. The present study therefore illustrated that methanogenic pathway shifting, induced by free ammonia, closely correlated to the process performance.