Objective: We conducted a dietary validation study in youth aged 1-11 years by comparing dietary intake of omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) as assessed by a parent-completed semiquantitative food frequency questionnaire (FFQ) over time to erythrocyte membrane composition of the same fatty acids. Design: The study population included youth aged 1-11 years who were participants in the Diabetes Autoimmunity Study in the Young (DAISY), a longitudinal study in Denver, Colorado that is following a cohort of youth at risk for developing type I diabetes. Four hundred and four children who had erythrocyte membrane fatty acid data matched to an FFQ corresponding to the same time frame for a total of 917 visits (matches) were included. PUFA intake was expressed both as g/day (adjusted for total energy) and as percent of total fat intake. We used mixed models to test the association and calculate the correlation between the erythrocyte membrane estimates and PUFA intake using all records of data for each youth. Results: Intakes of total omega-3 fatty acids (b ¼ 0.52, Po0.0001, r ¼ 0.23) and marine PUFAs (b ¼ 1.62, Po0.0001, r ¼ 0.42), as a percent of total fat in the diet, were associated with percent of omega-3 and marine PUFAs in the erythrocyte membrane. Intakes of omega-6 PUFAs (b ¼ 0.04, P ¼ 0.418, r ¼ 0.05) and arachidonic acid (b ¼ 0.31, P ¼ 0.774, r ¼ 0.01) were not associated. Conclusions: In these young children, an FFQ using parental report provided estimates of average long-term intakes of marine PUFAs that correlated well with their erythrocyte cell membrane fatty acid status.