Four parallel SBRs are established to treat synthetic wastewater with preset free ammonia (FA) concentrations (0.5, 5, 10 and 15 mg/L) named S0.5, S5, S10 and S15, respectively. The ammonium removal performance of the four systems both performed well throughout the experiment (average value of 98.7%). The inhibition of FA on nitrite-oxidizing bacteria (NOB) combined with process control used to achieve the nitrite pathway in S10 and S15 systems successfully. During initiating the nitrite pathway, the nitrite accumulation rate (NAR) dramatically increased to 90.3% on day 79 in S10 and 90.5% on day 139 in S15. In 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 plays an ever-increasing role in analyzing the relative abundance and structure of nitrifying bacteria in these samples. The results show that the changes in AOB and NOB abundance are consistent with our experimental results. FA not only affect the relative abundance of AOB and NOB but also the activity of NOB. Although AOB and NOB coexist in four systems, AOB is still the main nitrifying bacteria. Furthermore, we find a lower abundance of AOB had higher microbial utilization capacity of ammonia substrate at 15 mgFA/L.