We evaluated the accuracy of the estimated serving size using digital photographs in a newly developed food atlas. From 209 food items in the food atlas, we selected 14 items with various appearances for evaluation. At the study site, fifty-four participants aged 18–33 years served fourteen foods in the amount they usually ate. After they left, each food item was weighed by a researcher. The following day, the participants estimated the quantity of each food they served based on food photographs using a web-based questionnaire. We compared the weights of the foods the participants served (true serving sizes) and those determined based on the photographs (estimated serving sizes). For ten of the fourteen food items, significant differences were observed between the estimated and true serving sizes, ranging from a 29⋅8 % underestimation (curry sauce) to a 34⋅0 % overestimation (margarine). On average, the relative difference was 8⋅8 %. Overall, 51⋅6 % of the participants were within ±25 % of the true serving size, 81⋅9 % were within ±50 % and 93⋅4 % were within ±75 %. Bland–Altman plots showed wide limits of agreement and increased variances with larger serving sizes for most food items. Overall, no association was found between estimation errors and participant characteristics. The food atlas has shown potential for assessment of portion size estimation. Further development, refinement and testing are needed to improve the usefulness of the digital food photographic atlas as a portion size estimation aid.