The objective of this study was to compare the prevalence of risk factors for hepatitis C virus (HCV) infection among male prison inmates enrolling into a prospective cohort in Australia. We tested 121 inmates who were previously untested or were previously known to be anti-HCV antibody negative for anti-HCV antibodies by enzyme-linked immunosorbent assay. HCV-positive inmates were classified as cases (n = 25) and HCV-negative inmates as controls (n = 96). The study found that cases were less educated than controls and confirmed that prior imprisonment, drug injection, and a longer duration of injecting were risk factors for HCV infection. More than half of those who tested HCV positive perceived that they did not have HCV infection, and 44% were unsure of their HCV status. Those inmates who were incorrect about their HCV status tended to be less educated and were more likely to have been previously imprisoned than those who were correct about their HCV status. Inmates who were unsure of their HCV status were less likely to have been tested for HCV than those who had a clear perception of their HCV status, even if incorrect. Three (12%) inmates who tested positive denied injecting drug use, but reported other risk factors. Prisons are likely to remain an important site for the diagnosis of HCV infection and targeted interventions aimed at risk reduction among inmates with low education levels and a previous imprisonment history.