To reveal the shifts of microbial communities along ammonium gradients, and the relationship between microbial community composition and the anaerobic digestion performance using a high throughput sequencing technique. Methane production declined with increasing ammonium concentration, and was inhibited above 4 g l(-1). The volatile fatty acids, especially acetate, accumulated with elevated ammonium. Prokaryotic populations showed different responses to the ammonium concentration: Clostridium, Tepidimicrobium, Sporanaerobacter, Peptostreptococcus, Sarcina and Peptoniphilus showed good tolerance to ammonium ions. However, Syntrophomonas with poor tolerance to ammonium may be inhibited during anaerobic digestion. During methanogenesis, Methanosarcina was the dominant methanogen. Excessive ammonium inhibited methane production probably by decoupling the linkage between acidification process and methanogenesis, and finally resulted in different performance in anaerobic digestion.