Background The reproductive health and Prevention of Mother-to-Child Transmission of HIV (PMTCT) programs in Iran were integrated as a pilot project in September 2014.This study aims to provide a comprehensive evaluation and situation analysis of the PMTCT programme in Iran.Methods Pilot phase of PMTCT was launched in early September 2014 in selected centers including 170 health centers and 40 hospitals affiliated to medical universities of 16 provinces of Iran. In each medical university, a researcher-made checklist was administered to all newly-diagnosed HIV-positive pregnant women by the AIDS expert. Data was analyzed using SPSS 19 software.Results Overall, 69.4% of eligible pregnant women were enrolled in the pilot phase. From 134 reactive cases, 76 (56.7%) were confirmed as HIV positive. In 10 (13.2%) of HIV positive pregnant women, consumption of ARV was irregular. Also, 82.5% had CD4 count more than 350 after treatment with an average of 55.5% increase in the number of CD4 in comparison to the baseline, and 84.8% had viral load suppression (<200 copies/ml). Counseling and testing was done for the husbands of 75% of the women, and resulted in the identification of 15 (39.5%) new HIV cases among husbands. of tested individuals, 23 (60.5%) males already knew their HIV status and were registered HIV patients. HIV was diagnosed in one (1.5%) newborn.Conclusion Implementation of rapid HIV testing and PMTCT in Iran is strength in the national HIV control program. To eliminate MTCT, it is necessary to understand and overcome barriers and challenges the program faced in the pilot phase.