This study aimed to investigate hepatitis B virus (HBV) infection in military personnel in eastern China, which will provide a basis for the prevention of HBV infection.A total of 15,508 soldiers and 2386 officers were recruited from military camps in eastern China. The markers, deoxyribonucleic acid, serotypes, and genotypes of HBV in serum were detected and analyzed.Hepatitis B surface antigen (HBsAg) positive rate was 0.44% in soldiers, in whom the low-level HBsAg accounted for 88.24%. The HBsAg positive rate was 1.72% in officers in whom the low-level HBsAg accounted for 12.20%. There were significant differences in the prevalence of high-level and low-level HBsAg, HBV serotypes, HBV DNA positive rate, and mean log HBV DNA between officers and soldiers (P < .05). Compared with the conventional method for HBV DNA extraction, the enrichment method for HBV DNA extraction could significantly improve the positive rate and quantification of HBV DNA by real-time fluorescence quantitative polymerase chain reaction (P < .05). Sequencing of S gene in HBV was used for the determination of serotype and genotype of HBV. The sequencing success rate was significantly different between soldiers and officers (P < .05) as well as between high-level HBsAg group and low-level HBsAg group (P < .05). Significant difference was also observed in the genotype distribution between soldiers and officers (P < .05).HBV infection displays a low prevalence and a low epidemic state, and the prevalence of low-level HBsAg is higher in soldiers. We should pay attention to improve the quality of conscription examination as well as emphasize the surveillance, prevention, and protection of HBV infection in military officers.