Background and Study Aim Studies analyzed the extrahepatic reservoir of hepatitis B virus (HBV), especially in those with chronic HBV who are hepatitis B surface antigen positive but have a negative peripheral viremia, are still scarce. Therefore, we aimed to investigate the presence of HBV-DNA in peripheral blood mononuclear cells (PBMCs) and to evaluate different factors affecting this. Patients and Methods A total of 1650 naive chronic hepatitis B patients were recruited. Among these patients, 320 (19.4%) (75% were male [n = 240]; mean ± SD age, 38.4 ± 12.8 years) have a persistently negative serum real-time polymerase chain reaction (PCR) for HBV-DNA without previous treatment experience. For all patients, hepatic function tests and fibrosis assay by Fibroscan and hepatitis C virus coinfection, as well as HBV-DNA-PCR in both serum and PBMCs were analyzed. Results More than half of them (n = 170, 53.1%) exhibited positive HBV-DNA in PBMCs. The mean logarithm 10 of quantitative HBV-DNA by PCR in PBMCs was (5.1 ± 0.3 IU/mL). Hepatitis C virus coinfection was found in 30 patients (17.6%). Most of them (320 patients) had insignificant fibrosis scores (less than F2). The multivariate logistic regression analysis for prediction of presence of detectable HBV-positive viremia in PBMCs yielded the following risk factors (odds ratio [OR]): the presence of hepatitis C virus coinfection (OR = 1.7) and a logarithm 10 of quantitative hepatitis B surface antigen more than 3 (OR = 1.1). Conclusions A considerable number of patients with negative plasma HBV-DNA are still harboring subtle form of virus within remote extrahepatic compartments. Thus, dual testing for both plasma and PBMCs is mandatory especially in epidemiologic studies.