Summary Background The roles remain unclear of early on‐treatment quantitative serum HBsAg and hepatitis B virus (HBV) DNA levels in the prediction of a sustained response (SR) to peginterferon alfa‐2a therapy in HBeAg‐negative chronic hepatitis B (CHB) patients infected with genotype B or C. Aims To determine their roles in HBeAg‐negative CHB patients infected with genotype B or C. Methods Sixty‐one patients were treated with peginterferon alfa‐2a for 48 weeks. Serum HBsAg levels were quantified using the Abbott Architect HBsAg QT assay throughout treatment. Multiple regression analyses were performed to identify independent predictors of SR. Results Nineteen patients (31%) achieved SR with serum HBV DNA levels <312 copies/mL at 24 weeks post‐treatment. Serum HBsAg levels at 12 (OR 31.9; 95% CI 4.8–209.6; P = 0.0003) and 24 weeks of therapy (OR 8.8; 95% CI 2.0–38.0; P = 0.0035), and HBV DNA levels at baseline (OR 7.0; 95% CI 1.3–36.2; P = 0.0203), 12 (OR 7.9; 95% CI 1.2–48.4; P = 0.0249) and 24 weeks of therapy (OR 22.3; 95% CI 2.2–224.0; P = 0.0083) were early independent predictors of SR. A serum HBsAg cut‐off of 150 IU/mL at week 12 had an AUC, sensitivity, specificity and positive and negative predictive values of 0.75, 63%, 95%, 86% and 85% with respect to predicting SR respectively. Conclusions A quantitative serum HBsAg level at 12 weeks of therapy can be used for the early prediction of SR to peginterferon therapy in HBeAg‐negative CHB patients infected with genotype B or C.