A standardised, national, 160-item FFQ, the FFQ-NL 1.0, was recently developed for Dutch epidemiological studies. The objective was to validate the FFQ-NL 1.0 against multiple 24-h recalls (24hR) and recovery and concentration biomarkers. The FFQ-NL 1.0 was filled out by 383 participants (25-69 years) from the Nutrition Questionnaires plus study. For each participant, one to two urinary and blood samples and one to five (mean 27) telephone-based 24hR were available. Group-level bias, correlation coefficients, attenuation factors, de-attenuated correlation coefficients and ranking agreement were assessed. Compared with the 24hR, the FFQ-NL 1.0 estimated the intake of energy and macronutrients well. However, it underestimated intakes of SFA and trans-fatty acids and alcohol and overestimated intakes of most vitamins by >5 %. The median correlation coefficient was 039 for energy and macronutrients, 030 for micronutrients and 030 for food groups. The FFQ underestimated protein intake by an average of 16 % and K by 5 %, relative to their urinary recovery biomarkers. Attenuation factors were 044 and 046 for protein and K, respectively. Correlation coefficients were 043-047 between (fatty) fish intake and plasma EPA and DHA and 024-043 between fruit and vegetable intakes and plasma carotenoids. In conclusion, the overall validity of the newly developed FFQ-NL 1.0 was acceptable to good. The FFQ-NL 1.0 is well suited for future use within Dutch cohort studies among adults.