Early detection of Hepatitis B virus (HBV) in pregnant females starts by a request of the gynecologists, which is based on their knowledge and awareness of the guidelines on conducting these tests. This is an observational cross-sectional study that investigated the gynecologists’ knowledge, attitude and practice regarding HBV during pregnancy across Jordan. A random sample of 150 gynecologists were approached, from 3 major cities in Jordan, and asked to fill a questionnaire that assessed their knowledge, attitude and practice. Statistical analysis was conducted using SPSS. One hundred and seven gynecologists have participated in the study. Most of the respondents were females, residents, and less than thirty years old. Although 97.2% of the practitioners agreed on the importance of the pregnant females on HBV, only 43% were aware of the obligatory protocol in Jordan regarding HBV screening, and only 55% would screen the pregnant females to HBV in-practice. Significant association was found between screening rates to HBV and both, level of specialty and experience of practitioners. Approximately 60% of practitioners were aware of HBV perinatal transmission risk. Only 19.6% always referred the infected pregnant females to other specialists regardless of the viral load. While 47.7% of practitioners agreed on recommending antiviral therapy for third trimester pregnant females, only 12.1% would always/often prescribe them. A noteworthy lack of Hepatitis B knowledge and screening practice among gynecologists in Jordan have been observed. A national program designed to increase the awareness of HBV testing in Jordan for both patients and gynecologists is called for.