The present study reported the presence of a hepatitis B virus (HBV) major integration site (MIS) chr16: 51320015 and discussed the significance of quantitative measurement of this site. A total of 30 hepatitis B e antigen (HBeAg) positive (+) and 30 HBeAg negative (‑) patients with chronic hepatitis B (CHB) were enrolled in the present study, and the levels of intrahepatic (IH) covalently closed circular DNA (cccDNA), serum HBV DNA and hepatitis B surface antigen (HBsAg) were detected. Conventional reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and Sanger sequencing were designed to verify the chr16: 51320015 integration site, and the copy numbers of this site were measured using molecular clone and SYBR Green I RT‑qPCR. This site was found to be present in the hepatocytes of all the enrolled patients, and the average number of copies was 1.46x10‑2 ± 4.94x10‑2 copies/cell (3.48x10‑5‑0.212 copies/cell). No significant difference in the copy numbers of this site were observed between the HBeAg (+) (1.43 ± 9.79x10‑1 copies/cell) and HBeAg (‑) patients (6.58x10‑2 ± 2.47x10‑2 copies/cell; P>0.05), which were positively correlated with the levels of serum HBsAg (P=0.0038), but were not correlated with the levels of IH cccDNA (P=0.7785). In conclusion, the chr16:51320015 integration site may be a novel site, which persists in a several patients with HBV infection, and may accumulate in the hepatocytes due to clonal expansion. The diagnostic and therapeutic values of this site require further investigation.