Anaerobic digestion of chicken manure (CM) with high nitrogen content is easily suffered inhibition of ammonia, especially under thermophilic and high organic loading rate (OLR) condition. To screen the early warning indicators for instability, thermophilic digestion of CM was conducted in a bench-scale reactor (working volume 55 L) under OLR from 1.0 to 6.0 g VS/(L·d). Microbial community analysis was used to reveal the mechanism of instability at high OLR. The system collapsed at an OLR of 6.0 g VS/(L·d). The initial ammonia inhibition evoked serious acidification, subsequently, the dual suppression of free volatile fatty acids and free ammonia caused the final collapse. The most likely mechanism of iso-butyrate degradation was propionate pathway. The ratio of n-butyrate to iso-butyrate was screened as an early warning indicator with the threshold of above 0.45. The high OLR of 6.0 g VS/(L·d) broke the balance of syntrophic oxidation bacteria (norank_o__MBA03, Gelria, and norank_o__D8A-2), hydrogenotrophic methanogens (Methanothermobacter) and fermentative and acid-producing bacteria (Ruminiclostridium_1, Bacteroides, Defluviitalea, norank_o__Clostridia, and norank_o__M55-D21). The non-functional acid tolerant bacteria (Defluviitoga, norank_f__Family_XI, Corynebacterrium_1, Caldicoprobacter, Lactobacillus, and Erysipelothrix) became the dominant after the system collapsed.