Serum samples from the 1999 Mexico National Nutrition Survey (NNS) were analyzed to determine the prevalence of low serum B12 concentrations, identify factors related with low values including B12 intake, and importantly, to provide a baseline for monitoring progress in reducing deficiency. Samples for B12 were available from 488 children and 464 women, a sub-sample of the nationally representative 1999 NNS. The national overall prevalence of low (<200 pg/mL) and marginal (200 to 300 pg/mL) serum B12 was 25.6% and 21.0%, respectively. Adolescent girls had the lowest serum B12 concentrations (325 ± 308 pg/mL) and the prevalence of deficiency was 40% in pregnant women even using a lower cut-point (<135 pg/mL). Residents of rural areas and the South, population groups with poorest socioeconomic status, and illiterate and indigenous women had the lowest serum B12 Children and women who met dietary recommendations for B12 intake had higher serum B12 than those who did not. Overall 45.9% of intakes fell below the Estimated Adequate Requirement. Dietary B12 intake of children and women was directly correlated with serum B12 (r = 0.18, p < 0.001 and r = 0.11, p = 0.0304). The prevalence of marginal and deficient B12 status in 1999 was much higher than the most recently published national data suggesting the success of national policies to improve micronutrient status.