Background: The Mental Health Inventory-5 (MHI-5) is an internationally used screening measure for assessing mental health, with evidence supporting validity and reliability in different settings. So far, measurement invariance of the MHI-5, especially regarding gender, has rarely been investigated. Therefore, the aim of this study was to examine measurement invariance of the MHI-5 between men and women in a German population sample to enable valid and generalizable group comparisons. Methods: A total of 2075 participants (52.5% women) aged 18–64 years (M = 32.7, SD = 11.7) were proactively recruited at a municipal registry office in Germany and completed the MHI-5. The underlying factor structure was examined by comparing a one-factor model to a two-factor model. Multiple group confirmatory factor analysis was used to investigate measurement invariance between men and women. Results: A two-factor structure showed better model fit (χ²(4) = 106.388, Comparative Fit Index, CFI = 0.986) compared to a one-factor structure (χ²(5) = 193.652, CFI = 0.974). Constraining thresholds to equality did not lead to a significant loss of fit (χ²(10) = 9.370; p =.497, ΔCFI =.005). Further constraining loadings also did not reduce model fit (Δχ²(3) = 2.036; p =.565, ΔCFI =.001). Conclusion: The results demonstrated threshold and loading invariance for the MHI-5 between men and women. These findings suggest that the MHI-5 can be used for meaningful comparisons of latent means between men and women in the German population. Trial registration: German Clinical Trials Register (trial registration number: DRKS00014274, date of registration: 12 March 2018).