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This study aimed to assess changes in stress levels in the dental environment among students over a three-year period and to investigate associations with gender and academic level. A total of 149 students from a public university in southern Brazil completed the Dental Environment Stress (DES) questionnaire between 2020 and 2023, where higher scores reflect greater perceived stress. Stress evolution was evaluated using delta (Δ) scores (2023 minus 2020), meaning positive values indicate an increase in stress over time. Associations were tested by paired t-test and by adjusted linear regression. Most of participants were women (66.4%) and enrolled in undergraduate programs at baseline. Although women had higher absolute DES scores, men and undergraduate students showed greater increases over time. In addition to the total DES score, significant increases over time were observed in two specific domains: Patient Responsibilities (Δ = +3.94 ± 4.33 for men [p < 0.001] vs. +2.85 ± 4.95 for women [p < 0.001]) and Institutional Factors (Δ = +4.98 ± 6.18 for men [p < 0.0p1] vs. +2.26 ± 7.26 for women [p = 0.003]). In the adjusted models, men gender (β = 8.99; 95% confidence interval [CI]: 0.22-17.76) and undergraduate status (β = 13.36; 95% CI: 3.79-22.94) remained significantly associated with greater delta changes in DES scores. These findings suggest that the return to in-person clinical and academic activities after the pandemic may have affected students differently, depending on gender and academic level.