Assessing dietary intake in children is difficult and limited validated tools exist. Plasma carotenoids are nutritional biomarkers of fruit and vegetable intake and therefore suitable to validate reported dietary intakes. The aim of this study was to examine the comparative validity of a food frequency questionnaire (FFQ), completed by parents reporting child fruit and vegetable intake compared to plasma carotenoid concentrations. A sample of children aged 5–12 years (n = 93) from a range of weight categories were assessed. Dietary intake was measured using a 137-item semi-quantitative FFQ. Plasma carotenoids were measured using reverse phase high-performance liquid chromatography. Pearson correlation coefficients between reported dietary intake of carotenoids and plasma carotenoid concentrations were strongest after adjustment for BMI (β-carotene (r = 0.56, P < 0.05), α-carotene (r = 0.51, P < 0.001), cryptoxanthin (r = 0.32, P < 0.001)). Significantly lower levels (P < 0.05) of all plasma carotenoids, except lutein, were found among overweight and obese children when compared to healthy weight children. Parental report of children’s carotenoid intakes, using a FFQ can be used to provide a relative validation of fruit and vegetable intake. The lower plasma carotenoid concentrations found in overweight and obese children requires further investigation.