AbstractHigh and medium molecular weight of synthetic cationic polymers (3150 and 8265) and natural cationic polymer (chitosan) were selected to study the effect of cationic polymers on microbial granulation in anaerobic digestion. The optimal dose of each type of cationic polymer for the initial microbial agglomeration was determined in laboratory-scale jar test. The suitable dose of 3150, 8265 and chitosan was 2.0, 2.0 and 13.0mg/g VSS, respectively. The bench reactors were operated for a period of 120 days at organic loading rate (OLR) of 0.5-2.0kg COD/m3.d. As the results, these cationic polymers enhanced anaerobic granulation in term of granule properties which consists of physical, chemical and biological properties. Size distribution of all reactors increased specially addition of polymer 3150 which provided the granule percentage from 2.74% to 16.40%. Chitosan contributed on increased of extracellular polymeric substances (EPS) production from 20.2 to 50.0mg/g VSS, and also specific methanogenic activity (SMA) reached to 0.18g COD/g VSS.d. The experimental results showed that addition polymer 3150 and chitosan gave well productive in term of granule properties. Granule scanning electron microscope (SEM) and fluorescent in situ hybridization (FISH) with confocal laser scanning microscope (CLSM) photographs indicated the accomplishment of granulation by progressively increasing the OLR.