Background Suboptimal Health Status Questionnaire- 25 (SHSQ-25) is an established tool for measuring a precision health state between health and illness. The present study aims to assess the validity and reliability of a Persian version of SHSQ-25 (P-SHSQ-25) in a university staff Iranian population. Methods A sample of 316 academic and supporting staff (163 males, age range from 23 to 64 years old) from Hamadan University of Medical Sciences, Hamadan, Iran was recruited in this population- based cross-sectional study with a questionnaire validation from Apri1 to October 2022. Forward-backward translation method was performed for the SHSQ-25 translation from English to Persian. Internal reliability, content, convergence, discriminative and construct validity of the P-SHSQ-25 were examined. The factorial structure of the P-SHSQ-25 across groups was examined using measurement invariant test. Results In the translation process, the conceptual equivalence of the P-SHSQ-25 with the English version was confirmed. The item-content validity index and content validity ratio of all P-SHSQ-25 items were higher than the cut-off values of 0.70 and 0.62, respectively. Cronbach’s α was higher than 0.70 for all P-SHSQ-25 domains. The confirmatory factor analysis (CFA) showed the fitness of five factors on the data set (comparative fit index = 0.88, and root mean square error of approximation = 0.07). The CFA model fit did not change substantially across sex, age, occupation, economic status, and body mass index (Δ comparative fit index (CFI)<0.01). Conclusions The P-SHSQ-25 can be used as a reliable and valid tool to measure health status for screening pre-chronic disease conditions in a primary care setting among Iranian population.