This cross-sectional study evaluated knowledge and acceptability of prenatal diagnosis among 500 pregnant women at the University College Hospital, Ibadan. Most participants were aged 25-34 years, self-employed, Muslim, monogamy, secondary school leavers, on income of < naira10,000.00 (US$ 67.00)/month. Attitudinal mean score was dependent on age (p = 0.006), educational attainment (p = 0.001), marital status (p = 0.025) and religion (p = 0.012). Knowledge mean score was influenced by marital status (p = 0.028). Overall, acceptance of prenatal diagnosis was high. There was a direct correlation between acceptance and educational attainment: 41.5%, 31.50%, 19%, 19% of women who agreed to have prenatal diagnosis had tertiary, secondary school, primary school and no formal education respectively. Determinants of acceptability were age, educational attainment, marital status and religion. Being married significantly affected knowledge scores, while tertiary education, being divorced, unskilled and self-employed positively influenced attitude towards prenatal diagnosis.