Polymorphisms of maternal genes responsible for normal folate metabolism may be associated with an increased risk of fetal trisomy 21. By January 1998, most of Canada's flour was being fortified with folic acid. We investigated whether the prevalence of antenatally and postnatally detected trisomy 21 changed before and after folic acid food fortification. A total of 218,977 women underwent second trimester maternal serum screening for trisomy 21 in the 48 months before fortification and 117,986 women were screened in the 29 months after fortification. There were 375 identified cases of trisomy 21 before fortification (1.71 per 1,000), compared to 201 cases thereafter (1.70 per 1,000) for a crude prevalence ratio (PR) of 0.99 (95% confidence interval [CI] 0.84–1.18). The associated risk of trisomy 21 did not change after adjustment for mean maternal age (adjusted PR 0.99 [95% CI 0.82–1.19]). Similarly, no significant decline in the monthly prevalence of trisomy 21 was observed using autoregressive integrated moving average time series analysis (P = 0.24). In conclusion, we failed to observe a decline in the occurrence of trisomy 21 following folic acid food fortification. © 2003 Wiley-Liss, Inc.