Benzalkyl dimethylammonium compounds (BACs) are generally applied as surfactants and disinfectants. In this study, the nitrification systems were exposed to different alkyl chain lengths (C12-C16) and different levels of BACs (0-5 mg/L), respectively, totally 120 days and to explore the chronic effect of BACs on resistance genes (RGs). RGs were classified into four fractions based on activated sludge properties. Ammonia oxidation performance were not significantly affected by BACs, whereas BACs increased the absolute abundance of most intracellular RGs in sludge (si-RGs). Under the exposure of BACs, extracellular RGs in water (we-RGs) showed a decrease trend and si-RGs tended to be converted to we-RGs. Tightly bound-Tyrosine side chain was significantly correlated with most we-RGs, and we-intI1 might contribute to the propagation of RGs. Therefore, the risk of transmission of different fractions of RGs in the nitrification system under the stress of BACs should be taken seriously.