Quenching probe (QProbe) PCR-based methods for quantifying the abundance of Rhodocyclus-related and actinobacterial polyphosphate-accumulating organisms (PAOs) in an enhanced biological phosphorus removal (EBPR) process are described. PCR primer-QProbe sets specific for the 16S rRNA genes of Rhodocyclus-related PAOs (RPAOs) and actinobacterial PAOs (APAOs) were newly designed, and their specificity tested using database searches, a selection of pure cultures, and sludge DNA. The sets were found to be specific for most of the target sequences. QProbe PCR with the newly designed sets was used to enumerate the 16S rRNA genes of RPAOs and APAOs in a laboratory-scale EBPR sludge. The copy number of 16S rRNA genes of RPAOs was larger than that of APAOs before the phosphorus removal performance of the reactor deteriorated, and the amount of both PAOs decreased in the deterioration period. The approaches developed in this study may become a powerful tool for a high-throughput analysis of PAO abundance in EBPR processes.