The aim of this study was to investigate the prevalence of hepatitis B surface antigen (HBsAg) and clinical characteristics of middle school students infected with hepatitis B virus (HBV) after initiation of the HBV immunization program in China. A total of 82,156 serum samples were collected from students in 33 junior schools and 25 senior schools. HBsAg was tested by ELISA. Samples from HBsAg-positive students were collected and analyzed for HBV serum markers, alanine aminotransferase (ALT), HBV DNA levels, and HBV genotypes. The overall prevalence of HBsAg was 1.11% in middle school students in Shanghai, China. The prevalence of HBsAg in students born during the immunization program to HBsAg-positive mothers was significantly higher than that in students born during the universal vaccination program (1.47% vs 0.78%, P < 0.01). Only HBV genotypes B and C were found in these infections, and genotype C was the dominant one. Twenty-one (13.0%) of 162 HBsAg-positive students had active hepatitis B, and 18 were hepatitis B e antigen (HBeAg) positive. The universal infant vaccination program has reduced the prevalence of HBsAg significantly. HBeAg-positive hepatitis B, however, needs to be monitored among the students in whom vaccination failed.