Substantial percentage of HBV cases were reported among our population. Blood transfusion represent major rout transmission of Hepatitis B virus. The recent situation in Yemen increase the demand of blood and its products due to anemia, accidents, malnutrition, etc. The aim of the study was to estimate the current prevalence of HBsAg and total anti-HBc among blood donors in Sana’a, Yemen. A descriptive cross-sectional study was carried out at the National Blood Transfusion and Research Centre in Sana'a Yemen, during the period from October 2016 to January 2017. A total of 3606 blood donors were included in this study. Serum was separated from each blood specimens, and then they were analyzed for the presence of HBsAg, total Anti-HBc antibodies using an Enzyme Immunoassay technique according to manufacture instructions. The result of this study shown that, 4.1% and 14.0% donors were found to be reactive for HBsAg and anti-HBc respectively. The overall prevalence of HBV (HBsAg and/or anti-HBc positive) was 16.2%. According to the residency of the blood donors, a high percentage of HBsAg were observed among donors from Raymah 10.8% and Almahwet 10.3% governments followed by Alhudidah 7.0%. However, the lowest of HBsAg among blood donors coming from Capital city of Sana'a, Sana'a rural area and Taiz government was 3.9%. 3.6% and 3.5% respectively. In other hand, anti-HBc was found higher among donors from Raymah followed by Almahwet. Alhudidah and Albaidah governorates as 35.1%, 25.9%, 20.9%, and 20.0% respectively. This study concluded that our subjects still under the moderate to high-risk communities.