Objective:To analyse serum folate levels in women of childbearing age in the Metropolitan Region (MR) of Chile.Design:Cross-sectional design as part of the 2016-2017 National Health Survey (Encuesta Nacional de Salud, ENS 2016-2017), using a household-based multistage stratified random sample. Serum folate levels measured by electrochemiluminescence immunoassay in fasting venous blood samples were classified as deficient (<4·4 ng/ml), normal (4·4-20 ng/ml) or supraphysiological (>20 ng/ml).Setting:The MR of Chile.Participants:Women of reproductive age (15-49 years, n 222) from the MR participated in the ENS 2016-2017.Results:The mean, median and range of serum folate were 14·2 (se 0·4), 13·9 and 2·1-32·2 ng/ml, respectively. Folate deficiency was detected in 0·9 % of women, while 7·0 % had supraphysiological levels of the vitamin. No significant effects of age, educational level, marital status, parity, smoking status or nutritional status on serum folate levels were detected by univariate or multivariate analyses. Intake of folic acid supplements showed a significant association with serum folate levels, but only 1·2 % of women used supplements.Conclusions:Folate deficiency in women of reproductive age living in the MR of Chile is almost inexistent according to the ENS 2016-2017, suggesting that the current population-wide mandatory folic acid fortification of flour is an effective and equitable measure to prevent folate deficiency. These results support the option of maintaining current folic acid fortification in Chile, particularly based on the low adherence to supplementation regimes evidenced in other populations.