A standardised, national, 160-item FFQ, the FFQ-NL 1.0, was recently developed for Dutch epidemiological studies. The objective was tovalidate the FFQ-NL 1.0 against multiple 24-h recalls (24hR) and recovery and concentration biomarkers. The FFQ-NL 1.0 was filled out by383 participants (25–69 years) from the Nutrition Questionnaires plus study. For each participant, one to two urinary and blood samples andone to five (mean 2·7) telephone-based 24hR were available. Group-level bias, correlation coefficients, attenuation factors, de-attenuatedcorrelation coefficients and ranking agreement were assessed. Compared with the 24hR, the FFQ-NL 1.0 estimated the intake of energy andmacronutrients well. However, it underestimated intakes of SFA and trans-fatty acids and alcohol and overestimated intakes of most vitaminsby >5%. The median correlation coefficient was 0·39 for energy and macronutrients, 0·30 for micronutrients and 0·30 for food groups. TheFFQ underestimated protein intake by an average of 16% and K by 5 %, relative to their urinary recovery biomarkers. Attenuation factors were0·44 and 0·46 for protein and K, respectively. Correlation coefficients were 0·43–0·47 between (fatty) fish intake and plasma EPA and DHA and0·24–0·43 between fruit and vegetable intakes and plasma carotenoids. In conclusion, the overall validity of the newly developed FFQ-NL 1.0was acceptable to good. The FFQ-NL 1.0 is well suited for future use within Dutch cohort studies among adults.