Anaerobic ammonium-oxidizing (anammox) bacteria are well known for their aggregation ability. However, very little is known about cell surface physicochemical properties of anammox bacteria and thus their aggregation abilities have not been quantitatively evaluated yet. Here, we investigated the aggregation abilities of three different anammox bacterial species: “Candidatus Brocadia sinica”, “Ca. Jettenia caeni” and “Ca. Brocadia sapporoensis”. Planktonic free-living enrichment cultures of these three anammox species were harvested from the membrane bioreactors (MBRs). The physicochemical properties (e.g., contact angle, zeta potential, and surface thermodynamics) were analyzed for these anammox bacterial species and used in the extended DLVO theory to understand the force-distance relationship. In addition, their extracellular polymeric substances (EPSs) were characterized by X-ray photoelectron spectroscopy and nuclear magnetic resonance. The results revealed that the “Ca. B. sinica” cells have the most hydrophobic surface and less hydrophilic functional groups in EPS than other anammox strains, suggesting better aggregation capability. Furthermore, aggregate formation and anammox bacterial populations were monitored when planktonic free-living cells were cultured in up-flow column reactors under the same conditions. Rapid development of microbial aggregates was observed with the anammox bacterial population shifts to a dominance of “Ca. B. sinica” in all three reactors. The dominance of “Ca. B. sinica” could be explained by its better aggregation ability and the superior growth kinetic properties (higher growth rate and affinity to nitrite). The superior aggregation ability of “Ca. B. sinica” indicates significant advantages (efficient and rapid start-up of anammox reactors due to better biomass retention as granules and consequently stable performance) in wastewater treatment application.