Herpes simplex virus type 2 is the common cause of genital ulcer disease worldwide. Genital herpes infection is a major concern in pregnancy due to the risk of neonatal transmission. Materials and methods: A descriptive cross-sectional survey was conducted from August 2016 to March 2017 in some hospitals and health centers to assess the seroprevalence and risk factors of herpes simplex virus type 2 infection among pregnant women attending antenatal care in Dhamar city, Yemen. After taking written consent, socio-demographic, behavioral and obstetric history along with blood samples were collected from 200 pregnant women using a pre-structured questionnaire. Sera were analyzed for HSV-2 specific IgG using Electro-Chemiluminescence Immunoassay (ECLIA) method. Results: The overall seroprevalence of HSV-2 infection was 6 % (12/200) among pregnant women in Dhamar city. Levels of education and some obstetrical history such as numbers previous abortion (P=0.03), stillbirth (P=0.001). Although the study showed, there are significant differences in some symptoms, such as itching (P=0.02) and inflammation during urination (P=0.03). Conclusion: Overall, the seroprevalence of HSV-2 infection among pregnant women in Dhamar city is low. There is a critical need to adopt screening of HSV-2 into the antenatal profile tests. There is also need for more health education of this virus infection, methods of transmission, associated risk factors, and effective prevention and control strategies.