Background: Parvovirus B19 infection causes a wide range of complication in pregnant women including spontaneous abortion, severe fetal anaemia, non-immune hydrops fetalis and even intrauterine fetal death, erythema infectiosum (5th) disease and aplastic crisis. Objective: To perform molecular detection of human Parvovirus B19 among pregnant women attending antenatal clinic in Nnamdi Azikiwe Teaching Hospital Nnewi, Anambra State, Nigeria. Materials and Methods: This is a cross-sectional study conducted among 92 consented pregnant women, between the ages of 20-40 years who are attending antenatal clinic in Nnamdi Azikiwe University Teaching Hospital Anambra State, Nigeria, from May 2020-November 2020. A total of 92 blood specimen was collected from the pregnant women. Result: Out of the 20 IgM positive samples analyzed for Parvovirus B19 DNA, 13(65.0%) had Parvovirus B19 DNA and 7(35.0%) were negative for Parvovirus B19 DNA. Prevalence of Parvovirus B19 DNA in relation to gestational age was highest (69.2%) among pregnant women who were in their 2nd trimester and the prevalence was lowest (0.0%) among pregnant women who were in their 3rd trimester. Prevalence of Parvovirus B19 in relation to history of blood transfusion was highest (100.0%) among pregnant women who have had transfusion history and the prevalence was lowest (0.0%) among pregnant women who had no transfusion history. Conclusion: The frequency rate of Parvovirus B19 DNA seropostivity was highest (65.0%) among the pregnant women investigated. The association of Parvovirus B19 DNA seroposivity with gestation age and history of blood transfusion was statistically significant.