This study was aimed at comparing clinical applicability of serum HBsAg quantification in relation to intrahepatic covalently closed-circular DNA (cccDNA) in patients with HBeAg-positive and HBeAg-negative chronic hepatitis B (CHB) treated with pegylated interferon (PEG-IFN) monotherapy for 48 weeks. Overall, 32 and 36 patients with HBeAg-positive and HBeAg-negative CHB, respectively were recruited. Paired liver biopsies at baseline and end of therapy were analyzed for cccDNA. Virological response (VR) at 48 weeks post-treatment was defined as HBeAg clearance (for HBeAg-positive CHB) and HBV DNA <2,000 IU/ml (for both groups). The results demonstrated that baseline levels of all viral markers were higher in the HBeAg-positive group than the HBeAg-negative group. Baseline HBsAg correlated with cccDNA in the HBeAg-positive group (r = 0.452, P = 0.009) but not in the HBeAg-negative group (r = 0.018, P = 0.919). However, the magnitude of cccDNA and HBsAg decline at end of treatment was not different between groups. The reduction of HBsAg showed a positive correlation with cccDNA decline in HBeAg-positive and HBeAg-negative CHB (r = 0.544, P = 0.001 and r = 0.364, P = 0.029, respectively). Overall, responders had more decline in cccDNA and HBsAg levels compared with non-responders. Patients with serum HBsAg decline of >1.0 log10  IU/ml during treatment archived VR and HBsAg clearance of 80% and 30%, respectively. In conclusion, serum HBsAg represented a better surrogate marker of intrahepatic cccDNA in patients with HBeAg-positive CHB compared to those with HBeAg-negative CHB. On-treatment, HBsAg reduction of 1.0 log10  IU/mL was associated with a high probability of subsequent VR and HBsAg clearance in patients receiving PEG-IFN therapy. J. Med. Virol. 89:130-138, 2017. © 2016 Wiley Periodicals, Inc.