This study evaluated the validity and reliability of applying a newly developed dish-based, semi-quantitative food frequency questionnaire (FFQ) for Korean diet and cancer research. The subjects in the present study were 288 Korean adults over 30 years of age who had completed two FFQs and four 3-day diet records (DRs) from May 2008 to February 2009. Student’s t-tests, Chi-square tests, and Spearman’s rank correlation coefficients were used to estimate and compare intakes from different dietary assessment tools. Agreement in quintiles was calculated to validate agreement between the results of the second FFQ (FFQ-2) conducted in February 2009 and the DRs. Median Spearman’s correlation coefficients between the intake of nutrients and foods assessed by the FFQ-1 and FFQ-2 were 0.59 and 0.57, respectively, and the coefficients between the intake of nutrients and foods assessed by the FFQ-2 and the DRs were 0.31 and 0.29, respectively. The quintile classifications of same or adjacent quintile for intake of nutrients and foods were 64% and 65%, respectively. Misclassification into opposite quintiles occurred in less than 5% for all dietary factors. Thus this newly-developed, Korean dish-based FFQ demonstrated moderate correspondence with the four 3-day DRs. Its reliability and validity are comparable to those reported in other studies.