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Background: Food insecurity has a considerable impact on the social, physical, and psychological well-being of people and there is no food security assessment tool specific for Iranians. This study aimed to develop and assess the validity and reliability of an Iranian-specific food security questionnaire. Methods: The food security questionnaire was developed by five food security specialists by evaluating all available questionnaires (not specific to Iranians) in terms of applicability to Iranians. Furthermore, questions were developed from interviews conducted with ten families who were below the poverty threshold to understand how they described their food status. This questionnaire was administered to 200 households from different parts of Isfahan, Iran. Households were selected by multi-stage cluster randomized sampling. Households were categorized into 4 groups based on their score on the questionnaire; food secure (total score 0), mildly (total score 1-2), moderately (total score 3-7) and severely food insecure (total score 8-18). In the second stage of the study, 25 households were selected from each food security status group to evaluate the reliability and validity of the questionnaire by assessing sociodemographic, anthropometric, nutritional and biochemical parameters. Results: The prevalence of food security and mildly, moderately and severely food insecure were 24%, 33%, 27% and 16%, respectively. Content and face validity of the questionnaire was evaluated by experts, and latent class analysis confirmed construct validity. The developed questionnaire had good internal consistency (Cronbach's α = 0.91) and showed significant differences in hypothesized directions in food security status for sociodemographic factors. The prevalence of mothers, but not fathers or children, who had hemoglobin, mean corpuscular volume (MCV) and hematocrit less than the normal ranges increased (P = 0.04, P = 0.02, P = 0.02; respectively) with food insecurity. Conclusion: Our findings indicated that the developed questionnaire was a valid and reliable instrument to measure household food insecurity of Iranian families. [ABSTRACT FROM AUTHOR]