Four parallel SBRs were established to treat synthetic wastewater with preset concentrations of free ammonia (FA) (0.5, 5, 10 and 15 mg/L), including S_0.5, S₅, S₁₀ and S₁₅. The four systems removed ammonia well throughout the experiment (average value of 98.7%). The inhibition of FA by nitrite-oxidizing bacteria (NOB) combined with process control was used to achieve a nitrite pathway in S₁₀ and S₁₅. During the initiation of the nitrite pathway, the accumulation rate (NAR) increased dramatically to 90.3% on day 79 in S₁₀ and to 90.5% on day 139 in S₁₅. For S₁₀ on day 80~250 and S₁₅ on day 140~250, the average NARs were steady at approximately 98.8% and 98.2%, respectively. High-throughput sequencing of the 16S rRNA gene played an ever-increasing role in analyzing the relative abundance and structure of the nitrifying bacteria in these samples. The results showed that the changes in the abundance of AOB and NOB were consistent with our experimental results. FA affected not only the relative abundance of AOB and NOB, but also the activity of NOB. Although AOB and NOB coexisted in the four systems, AOB was still the main nitrifying bacteria. We found that a lower abundance of AOB had a higher microbial utilization capacity of ammonia substrate at 15 mgFA/L.