In 1998, Health Canada mandated folic acid fortification of white flour and enriched grain products to prevent neural tube defects. At the time, neither the Canadian Nutrient File (CNF) nor product labels reflected the actual folate content of foods. We aimed to assess if 20 years post-fortification, the CNF values for total folate and synthetic folic acid accurately reflect amounts determined by direct analysis. Using the 2001 Food Expenditure Survey and ACNielsen Company data, we identified 10 to 15 of the most purchased fortified foods across 7 food categories in Canada. Total folate concentrations were determined by tri-enzyme digestion and microbial assay. Folic acid concentrations were determined using liquid chromatography tandem mass spectrometry. Except for "cooked pastas", mean total folate content of foods (n=89) were significantly higher than CNF values across categories (p<0.05), reflecting 167% ± 54 of CNF values. Similarly, mean folic acid content of foods was higher than CNF values for all categories except "cooked pastas" (p<0.05), with a mean of 188% ± 94 of CNF values; the latter CNF values included uncooked pasta. In sum, 20 years post-fortification, and 10 years since the last direct measurement, CNF and product label values still underestimate actual total folate and the folic acid content of foods. These findings emphasize that dietary estimates established using the CNF may significantly underestimate actual intakes and thus caution should be exercised when interpreting estimates of nutritional adequacy based on these values.