Ammonia-oxidizing microorganisms (AOMs) play a crucial role in nitrogen removal in engineered systems. However, temperature fluctuations can lead to ammonia oxidation failure. This study investigated the effects of a broad temperature range (10.5, 14, 17.5, 21, 26.4, 35, 38.5, 42, and 45.5 °C) on the distribution of ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and comammox together with Nitrospira and Nitrobacter in gradient temperature-regulated reactors operated under low ammonia loading condition. Quantitative PCR and high throughput sequencing revealed that the AOA amoA gene numbers (∼10