Objective: To develop the Croatian version of the 41-item Dental Environment Stress questionnaire (DES) for stress assessment of dental students in both, preclinical and clinical years of study and to test its psychometric properties in Croatian dental student population. Materials and Methods: The English version of the 41-Item DES questionnaire was first translated into the Croatian language. Subsequently, it was set on the google drive and filled out by a total of 202 students from the School of Dental Medicine, University of Zagreb and 30 additional students from other Faculties. Students also assessed their overall level of stress on the Likert scale (1=no stress, 5=highest level of stress). Internal consistency was tested on 202 dental students; test-retest reliability on 30 dental students who filled out the same questionnaire twice; convergent validity on 202 dental students; and divergent validity on 202 dental students and 30 students from faculties not belonging to the biomedicine group. Results: Internal consistency showed high Cronbach alpha coefficient (0.9) and test-retest reliability showed no significant difference (P>0.05) within the period of 14 days when stress level had not changed (vacation). Convergent validity was confirmed by the significant association between the DES summary scores and the self- perceived level of stress (Spearman’s rho=0.881; P<0.001). Divergent validity was confirmed by significantly lower DES summary scores in students not belonging to the Biomedicine group (t=7.5, P<0.001). Conclusion: Excellent psychometric properties of the Croatian version of the DES questionnaire enable its utilization for assessment of stress level in Croatian dental students.