Background: Human immunodeficiency virus (HIV) testing of pregnant women and their partners is an important step in access to prevention of mother-to-child transmission (MTCT) services; pregnant women are at a high risk of seroconversion, which is associated with a high rate of MTCT. Repeat HIV testing in late pregnancy or in labor is worthwhile in areas with high HIV prevalence. This study aimed at determining HIV prevalence and seroconversion in a cohort of antenatal attendees at the Benue State University Teaching Hospital.Patients and Methods: This was a prospective cross-sectional study involving 432 cohorts of antenatal attendees. The HIV status of these women were determined at booking using rapid HIV kits: Determine ½, Stat-Pak, and Unigold Recombigen. Repeat testing was performed during labor among women who were HIV negative at booking to determine the seroconversion rate.Results: Overall HIV prevalence among this cohort of women was 8.6%. The seroconversion rate during labor was 2.5% among women who were seronegative at booking. Majority of the participants were young (modal age = 26–29 years, 36%), belonged to middle class (60.9%) and upper class (17.6%), and had secondary (50.0%) or tertiary education (38.0%).Conclusion: HIV prevalence and seroconversion rates are rather high. There is need for more aggressive PMTCT awareness advocacy and intervention to reduce the incidence of pediatric acquired immunodeficiency syndrome in our setting. Further studies to differentiate seroconversion from new infections in pregnancy are worthwhile PMTCT strategies.