Background: There is limited empirical data on the prevalence of Human Immunodeficiency Virus (HIV) seroconversion in late pregnancy due to the fact that majority of the women start seeking antenatal care late in pregnancy therefore rarely retest. This leaves a gap in the Elimination of Mother to Child Transmission (eMTCT) strategy thus contributing to high maternal and neonatal morbidity and mortality rates in Kenya. Objectives: To establish the rate of HIV retesting uptake in late pregnancy and HIV seroconversion rate among women attending Antenatal Clinics in Pumwani Maternity Hospital and Kakamega County Hospital. Methodology: A longitudinal study design was employed; 1,117 women confirmed as HIV negative in early pregnancy were followed up for three months and retested to ascertain their serostatus. Sample size was obtained through Census and consecutive sampling to recruit individual respondents. Clearance to conduct the study was sought from Ethics Review Committee, Pumwani and Kakamega County Hospitals and the respondents. Researcher administered questionnaires and HIV testing was done for data collection. Descriptive data involved calculation of measures of central tendency. Findings were presented in tables, graphs and narrative. Results: HIV retesting uptake was 1,073 (96%) and the rate of HIV serocopositivity among initially HIV negative pregnant women that retested was 8 (0.75%). Conclusion: HIV retesting uptake is very impressive and the rate of HIV seroconversion is very low. Kenya has made good progress towards eMTCT of HIV.