The deammonification process, which includes nitritation and anammox bacteria, is an energy-efficient nitrogen removal process. Starting up an anammox process in a wastewater treatment plant (WWTP) is still widely believed to require external seeding of anammox bacteria. To demonstrate the principle of a non-seeded anammox start-up, anammox bacteria in potential sources must be quantified. In this study, seven digesters, their substrates and reject water were sampled and quantitative polymerase chain reaction (qPCR) was used to quantify both total and viable anammox bacteria. The results show that mesophilic digesters fed with nitrifying sludge (with high sludge ages) can be classified as a reliable source of anammox bacteria. Sludge hygienization and dewatering of digestate reduce the amount of anammox bacteria by one to two orders of magnitude and can be considered as a sink. The sampled reject waters contained on average >4.0 × 104 copies mL−1 and the majority of these cells (>87%) were viable cells. Furthermore, plants with side-stream anammox treatment appear to have higher overall quantities of anammox bacteria than those without such treatment. The present study contributes to the development of sustainable strategies for both start-up of anammox reactors and the possibility of improving microbial management in WWTPs.