Background: A food frequency questionnaire (FFQ) for South American children and adolescents was developed, but its validity for assessing dietary iron intake has not been evaluated. Objective: To evaluate the validity of the FFQ and 24-hour dietary recalls (24h-DR) for assessing dietary iron intake in children and adolescents. Design: The South American Youth/Child Cardiovascular and Environmental study is a multicenter observational study, conducted in five South American cities: Buenos Aires (Argentina), Lima (Peru), Medellin (Colombia), Sao Paulo, and Teresina (Brazil). The FFQ assessed dietary intake over the previous 3 months, and the 24h-DR was completed three times (2 weekdays and 1 weekend day) with a minimum 5-day interval between recalls. Blood samples were collected to assess serum iron, ferritin, and hemoglobin levels. Participants and setting: Data of 99 children (aged 3 to 10 years) and 50 adolescents (aged 11 to 17 years) from public and private schools were collected during 2015 to 2017. Main outcome measures: Dietary iron intake calculated from the FFQ (using the sum of daily iron intake in all food/food groups) and 24h-DR (mean of 3 days using the multiple source method). Statistical analyses performed: Dietary iron intake in relation to blood biomarkers were assessed using Spearman rank correlations adjusted for sex, age, and total energy intake, and the quadratic weighted κ coefficients for agreement. Results: Spearman correlations showed very good coefficients (range = 0.78 to 0.85) for the FFQ in both age groups; for the 24h-DR, the coefficients were weak in children and adolescents (range = 0.23 to 0.28). The agreement ranged from 59.9% to 72.9% for the FFQ and from 63.9% to 81.9% for the 24h-DR. Conclusion: The South American Youth/Child Cardiovascular and Environmental study FFQ exhibited good validity to rank total dietary iron intake in children and adolescents, and as well as the 24h-DR, presented good strength of agreements when compared with serum iron and ferritin levels.