The Depression Anxiety Stress Scales-21 (DASS-21) is a commonly used scale to briefly assess psychological distress in both research and clinical practice. Although the DASS-21 has been validated for use in numerous cultural, racial, and cisgender populations, its invariance for transgender and nonbinary (TGNB) populations has yet to be tested. Considering prior findings of invariance for similar measures of depression and anxiety with TGNB samples, it is important to understand whether scores for the DASS-21 differ as a function of gender identity. Using a sample of 546 (52.5%) cisgender sexual minority and 494 (47.5%) TGNB participants (N = 1040), configural, metric, and scalar invariance were tested through multiple group confirmatory factor analysis (CFA) for both cisgender versus broad TGNB participants as well as within TGNB identity groups. The DASS-21 demonstrated all levels of invariance for both groupings, suggesting that the factor structure, factor loadings, and item intercepts are equal across cisgender and TGNB participants and within TGNB identities. These results indicate that the DASS-21 can be used for TGNB populations to assess their level of psychological distress and that past studies which used this measure to understand levels of psychological distress among TGNB individuals were not biased by measurement error. Public Significance Statement The present study supports the use of the Depression Anxiety Stress Scales-21 (DASS-21) to assess levels of psychological distress among transgender and nonbinary (TGNB) individuals. Further, the results indicate that studies which seek to compare rates of psychological distress between cisgender and TGNB individuals should consider using the DASS-21 because the measure was shown to perform similarly for these populations.