The association between temperament and drug use or temperament and psychopathology has previously been restricted to community clinical or non-clinical samples. The aim of the present study was therefore to examine these associations in a large cohort of Australian offenders using Cloninger's Temperament and Character Inventory (TCI). A total of 1322 prisoners from New South Wales (NSW) who completed all dimensions of the TCI and were screened for mental illness using the Australian National Survey of Mental Health and Wellbeing were included in the study. A total of 15% of the sample fulfilled the criteria for a diagnosis for depression, 36% for anxiety disorders and 54% for a substance abuse disorder. Using logistic regression analysis, the TCI dimensions of harm-avoidance and low self-direction predicted depression. Being female, a poly-substance user and having high harm-avoidance, persistence, self-direction and self-transcendence predicted anxiety disorders. Significant stepped trends across age, gender, and type of drug use were found for all TCI dimensions. The TCI is useful in identifying prisoners with a history of psychopathology and substance misuse. This tool also provides clinically relevant information about at-risk individuals and has the potential to guide the development of intervention programmes for inmates.