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Zika virus (ZKV) infection in a pregnant woman, especially during the first trimester, often results in congenital anomalies. However, the pathogenic mechanism is unknown and one-third of ZKV infected pregnancies are asymptomatic. Neutralizing antibodies against ZKV has been reported in 70% of Thai adults, but the prevalence among pregnant women is unknown. Currently, vaccines and specific treatments for ZKV are under development. A better understanding of the immune status of pregnant women will increase the success of effective prevention guidelines. The prevalence of ZKV infection in pregnant women in antenatal care clinics was investigated during the rainy season from May to October 2019 at Siriraj Hospital, Bangkok, Thailand. We recruited 650 pregnant women (39.42% first, 52.26% second and 7.36% third trimester) and found that 30.77% had ZKV-specific IgG, and 39.81% had neutralizing antibodies (nAb) against ZKV (titer ≥10). Specific and neutralizing antibody levels varied by maternal age, trimester, and month. We further characterized the cross-reaction between ZKV and the four Dengue virus (DENV) serotypes by focused reduction neutralization test (FRNT) and found that cross-reactions were common. In conclusion, about 60% of pregnant women who living in central Thailand may be at risk of ZKV infection due to the absence of neutralizing antibodies against ZKV. The functions of cross-reactive antibodies between related viral genotypes require further study. These findings have implications for health care monitoring in pregnant women including determining the risk of ZKV infection, assisting the development of a flavivirus vaccine, and informing the development of preventative health policies.