The aims of this prospective study were to examine the variation of dental students' perceived stressors throughout the course of their dental studies and to explore the role of gender on stress. A thirty-item modified version of the Dental Environment Stress Questionnaire (DES) was administered annually between 2003 and 2007 to a cohort of 109 Greek dental students enrolled in a five-year D.D.S. curriculum. Descriptive and multivariate longitudinal methods were utilized to examine variations in perceived stressors by year of study and by gender. Response rates ranged from 61 to 97 percent. Mean scores for the DES factors "workload" and "clinical training" decreased over the study period, while "faculty and administration" increased. Males reported substantially lower perceived stress in the "self-efficacy beliefs" (adjusted DES difference=-0.40; 95 percent CI=-0.63, -0.16), "workload," and "performance pressure" domains. Longitudinal analysis revealed substantial changes for numerous individual stressors. Most concerns for "examinations and grades" were expressed in year three (OR=2.7; 95 percent CI=1.4, 5.0; reference: first year), whereas "lack of self-confidence" items peaked in the second year. Marked gender differences in stress appraisal persisted over time. Longitudinal changes in dental students' stress perceptions corresponded with transitions in the didactic, preclinical, and clinical phases of the curriculum.