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Background The reproductive health and Prevention of Mother-to-Child Transmission (PMTCT) of HIV programs in Iran were integrated as a pilot project in September 2014. This study aims to provide a comprehensive evaluation and analysis of the PMTCT of HIV program in Iran. Methods The pilot phase of PMTCT of HIV 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 an AIDS expert. Data was analyzed using SPSS 19. 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. ARV consumption was irregular in 10 (13.2%) of HIV positive pregnant women. 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 that resulted in the identification of 15 (39.5%) new HIV cases among husbands. Among the tested individuals, 23 (60.5%) males already knew their HIV status and were registered as HIV patients. HIV was diagnosed in one (1.5%) newborn. Conclusion Implementation of rapid HIV testing and PMTCT in Iran is one of the strengths of the national HIV control program. To eliminate MTCT, it is necessary to understand and overcome the barriers and challenges to the program in the pilot phase.