Objective: This study aimed to assess the validity and reliability of a dish-based, semi-quantitative food frequency questionnaire (DFFQ) for epidemiological studies in Iran. The DFFQ included 142 items (84 foods and 58 mixed dishes) which was filled in by 230 adults (110 men). All participants completed two separate DFFQs with a 6 months interval as well as six 24-h recalls, each month. Dietary biomarkers and anthropometric measurements were made. The validity was evaluated by comparing the DFFQ against 24-h dietary recalls and dietary biomarkers, including serum retinol and beta-carotene. Reliability was evaluated using intra-class correlation coefficient (ICC) and validity was determined by unadjusted and energy adjusted correlation coefficients (CC), de-attenuated CC, and cross-classification analyses. Results: ICC for reliability ranged between 0.42 and 0.76. De-attenuated CC for the FFQ and the 24-h recalls ranged between 0.13 and 0.54 (Mean = 0.38). The de-attenuated CC between the DFFQ and plasma levels of retinol and beta-carotene were 0.58 (P = 0.0001) and 0.40 (P = 0.0001), respectively. Cross-classification analysis revealed that on average 73% were correctly classified into same or adjacent quartiles and 5% were classified in opposite quartiles.