Introduction: Nowadays, HIV is mostly spreading in Asian countries. One of the important routes for HIV transmission in these countries is the vertical route which infects 35% to 45% of newborns. Mother’s education, drug prophylaxis and Cesarean section, accompanied by banning breastfeeding will decrease this rate to 2%. Therefore, mothers’ knowledge about Prevention of Mother to Child Transmission (PMTCT) has a great role in HIV/AIDS prevention. This study was designed to evaluate knowledge of pregnant women about HIV, its vertical transmission and prevention in Tehran, Iran.Materials and Methods: This cross-sectional study was conducted on 1577 pregnant women aged 15 - 46 years who were attending prenatal care clinics in Tehran, Iran. The research material was a questionnaire which was completed daily by trained midwives. The data were statistically analyzed by ANOVA, independent sample t-test, Pearson correlation and linear regression with a significance level of p = 0.05.Results: About 16.5% of the participants had good knowledge about HIV/AIDS and 54.1% about its transmission routes but awareness about its prevention was only 5.7%. Fifty-seven percent of the participant had not been tested for HIV earlier and 20.2% were not willing to undergo such tests. About 86.2% of the participants had no idea about the availability of drug prophylaxis in Iran for PMTCT.Conclusion: The fact that 28.2% of the participants were not willing to undergo HIV testing reflects negative attitude about HIV infection. Although the overall awareness about the infection and its transmission was good but knowledge about its prevention especially by PMTCT and its availability in Iran was low. Educational programs through mass media or prenatal care programs by focusing on HIV/AIDS prevention maybe useful.