To explore the feasibility of the rapid start-up of partial denitrification and the stable accumulation of NO2--N in different waste sludge sources, three identical SBR reactors (S1, S2, and S3) were inoculated respectively with sludge discharged from a laboratory municipal wastewater denitrifying phosphorus removal system, surplus sludge from a municipal wastewater treatment plant, and river sediment sludge. The characteristics of the partial denitrification start-up and NO2--N accumulation were compared, and the partial denitrification activity of the system or NO3--N→NO2--N transformation performance were investigated by analyzing the characteristics of the functional bacteria genera of the reactor from the perspective of microbiology. The results showed that all three SBR partial denitrification reactors could be launched successfully in a short time with sodium acetate as the sole carbon source, under a high alkalinity, and by using a suitable COD/NO3--N ratio. The average NO3--N→NO2--N transformation ratio of the system was ranked as:S1 > S2 > S3 (75.92% > 73.36% > 69.90%). It was found that S1 and S2 had different degrees of partial denitrification performance deterioration under a continuous low temperature, but that S3 could maintain a good NO2--N accumulation performance. High throughput sequencing showed that Proteobacteria and Bacteroidetes were dominant in the partial denitrification system, and that the abundance of Thauera was significantly different in the three PD reactors:S3 > S1 > S2 (25.09% > 4.71% > 3.60%), thus indicating that S3 had stable and efficient NO2--N accumulation performance and that a high abundance of Thauera might play a significant role in maintaining low temperature partial denitrification activity.