To establish efficient methods for self-prevention of oral diseases, assessment of dental health behavior and knowledge in various social classes is necessary. The main purpose of this study was to determine the major differences in oral health knowledge and behavior in a group of Iranian pre-university students. In this cross-sectional study, 591 pre-university students from different regions of Mashhad, Iran were randomly selected to complete a questionnaire consisting of two parts including dental health behavior and knowledge. Scores were recorded and statistical analyses performed to determine the correlation between dental health behavior and knowledge. Data was analyzed with Student's t-test, one-way analysis of variance and Pearson correlation. The mean score of dental health knowledge was significantly lower than the dental health behavior (2.95 ± 0.02 vs. 3.31 ± 0.05, P < 0.001). This difference was observed with gender, birth location and major subject of study. The dental health behavior of Iranian pre-university students was inadequate and their dental health knowledge was at a lower level compared to their behavior. Experimental science students had better oral health behavior compared to other students. (J Oral Sci 53, 177-184, 2011)