Hepatitis B infection caused by hepatitis B virus (HBV) is one of the most important public health threats worldwide. HBV infects the liver and can drive serious liver complications which may lead to death in most cases. In 2007, viral hepatitis was classified by Saudi Ministry of Health as the second most common viral disease with 9000 new cases and HBV represented 52% of all diseases. The prevalence rate of HBV in Saudi Arabia in 2019 was 14.58 per 100.000 population. However, in some cases, HBsAg, appears as negative, which is known as occult HBV infection (OBI). OBI can be defined as the presence of HBV DNA in liver, while HBsAg is negative in serum. Therefore, this study aimed to investigate the rate of OBI in premarital screening (PMS) serum samples, which can be a hidden mode of spread of this disease, and raise awareness about it. A cross-sectional study was conducted in three tertiary level public hospitals in Buraydah City, Al-Qassim, Saudi Arabia within the period of September 2018 to January 2019. A total of 848, HBs Ag Negative Premarital screening serum samples, were randomly collected from both genders, attending the three different hospitals under the Premarital Screening program in Qassim region. All serum samples were then tested by ELISA technique to detect viral antigen and antibodies, and DNA viral load was then detected using Q-PCR. Out of 848 HBs Ag negative serum samples, only 28 samples were found to be reactive to anti-HBc Ab, while 820 samples were non-reactive to anti-HBc Ab. Among the anti-HBc Ab reactive samples, 24 samples were also reactive to anti-HBs Ab, while only 4 samples were, anti-HBs Ab non-reactive. Furthermore, these 4 samples were found to be below detecting range, following analysis of HBV DNA using Q-PCR. This study showed that the probable, prevalence rate of OBI in Qassim region was 3.30%, with no differences among gender or age groups. Therefore, it is highly recommended to introduce anti-HBc assay screening, as a criteria under the Premarital Screening Program. More studies are required to focus on OBI and conduct further investigations with consideration of family history.