Objectives: Hepatitis B virus (HBV) infection is a global health burden. In this regard, Egypt has an intermediate HBV seroprevalence. HBV is classified into ten different genotypes (A-J) with different geographic distributions. Genotype D is the most prevalent in the Middle East. Limited data are available about HBV genotyping among Egyptian blood donors, particularly in Upper Egypt. We examined the seroprevalence of HBV among 12,000 blood donors attending the blood transfusion services center in Minia Governorate, Upper Egypt. Methods: HBsAg and HBeAg were examined by ELISA while HBV-DNA was examined by PCR. HBV genotyping was conducted by restriction fragment length polymorphism. Results: HBsAg was detected in 237 donors (1.98%). The HBV-DNA of 50 donors with the highest HBsAg OD was examined for the HBV genotype. All 50 DNA-positive samples were of genotype D. 82% of the DNA-positive donors were males, coinciding with their representation in the cohort. ALT levels were normal in 88% of genotyped subjects, while 84% of them were HBeAg negative. Conclusion: Taken together, these data indicate that HBV genotype D is the predominant genotype among HBsAg-positive blood donors in Upper Egypt and was in >80% of the subjects associated with a negative HBeAg serostatus.