Vitamin B12 deficiency may be an independent risk factor for neural tube defects (NTD). We determined the prevalence of biochemical B12 deficiency (<125 pmol/l) among 10 622 Ontarian women aged 15-46 years who underwent concomitant testing of serum bhCG and B12 9 years after the implementation of Canadian folic acid flour fortification. The overall prevalence of biochemical B12 deficiency was 7.4%. Relative to non-pregnant women, the adjusted odds ratio (95% confidence interval) of biochemical B12 deficiency was 0.78 (0.60-1.0) among women pregnant 28 days gestation or less and was 1.4 (1.1-1.8) after 28 days gestation. About 1 in 20 women may be deficient in B12 in early pregnancy. The impact on maternal and fetal well-being, including preventable NTD, should be considered.