Phylogenetically diverse anammox bacteria have been detected in most of anoxic natural and engineered ecosystems and thus regarded as key players in the global nitrogen cycle. However, ecological niche differentiation of anammox bacteria remains unresolved despite its ecological and practical importance. In this study, the microbial competitions for a common substrate (nitrite) among three anammox species (i.e. “Candidatus Brocadia sinica”, “Candidatus Jettenia caeni” and “Candidatus Kuenenia stuttgartiensis”) were systematically investigated in nitrite-limited gel-immobilized column reactors (GICR) and membrane bioreactors (MBRs) under different nitrogen loading rates (NLRs). 16 S rRNA gene-based population dynamics revealed that “Ca. J. caeni” could proliferate only at low NLRs, whereas “Ca. B. sinica” outcompeted other two species at higher NLRs in both types of reactors. Furthermore, FISH analysis revealed that “Ca. J. caeni” was mainly present as spherical microclusters at the inner part (low NO2− environment), whereas “Ca. B. sinica” was present throughout the gel beads and granules. This spatial distribution supports the outcomes of the competition experiments. However, the successful competition of “Ca. J. caeni” at low NLR could not be explained with the Monod model probably due to inaccuracy of kinetic parameters such as half saturation constant (Ks) for nitrite and a difference in the maintenance rate (m). In addition, the growth of “Ca. K. stuttgartiensis” could not be observed in any experimental conditions, suggesting possible unknown factor(s) is missing. Taken together, NLR was one of factors determining ecological niche differentiation of “Ca. B. sinica” and “Ca. J. caeni”.