Background: Infection with herpes simplex virus is one of the most common sexually transmitted infections. Because the infection is common in women of reproductive age it can be contracted and transmitted to the fetus during pregnancy and the newborn, whichcan lead to death or long-term disabilities. Objectives: The aim of our study was todetect the seroprevalence of Herpes Simplex Virus infection in pregnant females at Sohag University Hospital using Enzyme Linked Immunosorbent Assay (ELISA)confirmed by Polymerase Chain Reaction (PCR) as well as the fetal outcome of infectedwomen in comparison with healthy. Methodology: The study was carried out on 60pregnant women attending the Obestetric and Gynacology department of SohagUniversity Hospital fulfilled the inclusion criteria. They were screened for type specificHSV antibodies (HSV-1 IgG and IgM, HSV-2 IgG and IgM) using Enzyme LinkedImmunosorbent Assay (ELISA). For IgM positive cases, HSV DNA was detected byPolymerase Chain Reaction (PCR). Blood samples were collected from all theparticipants after written informed consent. Univariate and multivariate analysis wereperformed to identify the risk factors associated with HSV positivity. Results: ELISA testfor HSV-1 antibodies revealed 14 (23.33%) cases positive for IgM and 44 (73.33%)cases positive for IgG antibodies. For HSV-2 antibodies there were 4 (6.67%) casespositive for IgM and 38 (63.33%) cases positive for IgG antibodies. By PCR HSV-DNAwas detected in 14 (77.77%) of the 18 ELISA positive cases representing (23.33%) of thestudied populations. Conclusion: According to our study overall prevalence of HSVinfection among pregnant women in Sohag University Hospital is about 23%, mostly dueto HSV-1.