Background: Differences in habitual dietary fiber intake may modify effects of dietary fiber interventions, thus measurement of habitual dietary fiber intake is relevant to apply in intervention studies on fiber-rich foods, and food frequency questionnaire (FFQ) is a commonly used method. Rye bread is the major contributor of dietary fiber in the Danish population, and a nation-specific FFQ is therefore needed. Objective: The aim of this study was to assess the relative validity and reproducibility of a self-administered quantitative FFQ designed to assess total dietary fiber intake among Danish adults. Design: In order to assess the relative validity of the FFQ, a total of 125 participants completed both a 7-day weighed dietary recording (DR) and an FFQ consisting of 60 questions. To evaluate the reproducibility of the FFQ, a sub-group of 12 participants subsequently completed an FFQ approximately 6 months later. Results: Estimates of mean dietary fiber intake were 24.9±9.8 and 28.1±9.4 g/day when applying the FFQ and DR, respectively, where FFQ estimates were ~12% lower (p<0.001). Pearson's correlation coefficient between the estimated dietary fiber intake of the two methods was r=0.63 (p<0.001), and 62% of the participants were grouped into the same tertile of intake according to the two methods. The estimates of mean dietary intake of first and second FFQ were very similar (22.2±4.0 and 23.3±4.1 g/day, respectively, p=0.42) and showed a correlation of r=0.95 (95% CI 0.83–0.99). Conclusion: The developed FFQ showed moderate underestimation of dietary fiber intake (g/day), adequate ranking of subjects according to their dietary fiber intake, and good reproducibility. The FFQ is therefore believed to be a valuable tool for epidemiology and screening in human interventions, where intake of dietary fibers is of specific interest.