Membrane bioreactors (MBR) are an important and increasingly implemented wastewater treatment technology, which are operated at low food to microorganism ratios (F/M) and retain slow-growing organisms. Enhanced biological phosphorus removal (EBPR)-related organisms grow slower than ordinary heterotrophs, but have never been studied in detail in MBRs. This study presents a comprehensive analysis of the microorganisms involved in EBPR in pilot- and full-scale MBRs, using fluorescence in situ hybridization (FISH), as well as an overall assessment of other relevant microbial groups. The results showed that polyphosphate accumulating organisms (PAOs) were present at similar levels in all studied MBRs (10% ± 6%), even those without a defined anaerobic zone. Glycogen accumulating organisms were also detected, although rarely. The FISH results correlated well with the observed P removal performance of each plant. The results from this study suggest that a defined anaerobic zone is not necessarily required for putative PAO growth in MBRs, since polyphosphate storage may provide a selective advantage in fulfilling cell maintenance requirements in substrate-limited conditions (low F/M).