The DASS-21 is a public domain instrument that is commonly used to evaluate depression and anxiety in psychiatric and community populations; however, the factor structure of the measure has not previously been examined in oncologic settings. Given that the psychometric properties of measures of distress may be compromised in the context of symptoms related to cancer and its treatment, the present study evaluated the psychometric properties of the DASS-21 Depression and Anxiety scales in cancer patients (n = 376) as compared to noncancer control participants (n = 207). Cancer patients ranged in age from 21 to 84 years (mean = 58.3, standard deviation = 10.4) and noncancer control participants ranged in age from 18 to 81 years (mean = 45.0, standard deviation = 11.7). Multiple group confirmatory factor analysis supported the structural invariance of the DASS-21 Depression and Anxiety scales across groups; the factor variance/covariance invariance model was the best fit to the data. Cronbach’s coefficient alpha values demonstrated acceptable internal consistency reliability across the total sample as well as within subgroups of cancer patients and noncancer control participants. Expected relationships of DASS-21 Depression and Anxiety scale scores to measures of suicidal ideation, quality of life, self-rated health, and depressed mood supported construct validity. These results support the psychometric properties of the DASS-21 Depression and Anxiety scales when measuring psychological distress in cancer patients.