Biological nutrient removal (BNR) plants can reduce both carbon and oxygen requirements by increasing the fraction of phosphorus (P) removed by denitrifying polyphosphate accumulating organisms (DPAOs). Contrasting findings have been reported in literature concerning whether or not PAOs and DPAOs are different microorganisms. In this study, quantitative fluorescence in situ hybridisation (FISH) measurements from different EBPR sludges support the hypothesis that PAOs and DPAOs are phyogenetically different. This experimental evidence is discussed within the context of literature findings and suggestions for future research concerning the identity of PAOs and DPAOs are proposed. Further, this paper discusses the different methodologies available for assessing the DPAO fraction through chemical analytical techniques, where the relative fraction estimated is highly dependent on the methodology employed. Thus, we recommend an alteration to previously proposed methods in order to calculate the DPAO fraction through anaerobic-anoxic and anaerobic-aerobic batch tests. This information is expected to be valuable in studies focussed on optimising the amount of phosphorus removal achieved with simultaneous denitrification.