Psychological science faces a call to action researching the implications of the corona virus disease 2019 (Covid-19) pandemic. Rapid reviews have reported that maintaining rigorous research standards is a priority for the field, such as ensuring reliable and valid measurement, when investigating people’s experience of Covid-19 (O’Connor et al., 2020). However, no research to date has validated a measure mental health symptomology for an athlete population. The current research addresses this gap by examining the internal consistency, factor structure, invariance, and convergent validity of the Depression Anxiety and Stress Scale (DASS-21; Lovibond and Lovibond, 1995) in two athlete samples. Participants completed the DASS-21 and sport-specific measures of mental health such as the Profile of Mood States – Depression subscale (POMS-D), Sport Anxiety Scale-2 (SAS-2), Athlete Burnout Questionnaire (ABQ), and Athlete Psychological Strain Questionnaire (APSQ). In sample one (n = 894), results of exploratory structural equation modeling indicated that a three-factor model provided good fit to the data, but a bifactor model provided better fit. Factor loadings indicated minimal misspecification and higher loadings on the general-factor. Invariance testing suggested equivalence across gender, athletic expertise, sport type, and injury status. Further, latent mean differences analyses indicated that females and injured athletes scored higher than male and non-injured athletes on all DASS-21 factors reporting higher mental health symptomology, those with more expertise scored higher on the general-factor and depression and those with less expertise scored higher on anxiety and stress, and no differences between team and individual athletes. In sample two (n = 589), the bifactor structure was replicated. Results largely supported the scales convergent validity with depression predicting POMS-D scores, whereas all three subscales predicted the SAS-2, ABQ, and APSQ scores. Internal consistency was acceptable in both samples. The current work provides initial support for use of the DASS-21 as an operationalisation of mental health symptomology in athletes. Theoretical and practical implications of these results are discussed.