High hepatitis C virus (HCV) prevalence has been documented among many injecting drug user (IDU) populations worldwide; however, there is limited published data on trends in incidence of infection in these epidemics over time. To address this, we used a novel method of analyzing data collected via repeat, cross-sectional sero-surveys by injection initiation cohorts to investigate trends in HCV seropositivity among a population of needle and syringe program (NSP) attendees in Australia between 1995 and 2004, and thereby infer annual incidence trends. Injection initiation cohorts were defined by their time of entry into the IDU population. We also investigated the associations between HCV antibody seroprevalence and risk factor data, and trends in risk factor data over the decade. Approximately 20,000 NSP attendees participated in the study over the 10-year period. Within each injection initiation cohort, we found an increase in HCV prevalence over time, with prevalence appearing to reach saturation around 90%. There was little indication that the slopes of increase had changed with more recent initiation cohorts. While duration of injecting was most strongly associated with HCV seropositivity in this study, we also found that self-reported history of needle and syringe sharing and imprisonment were independently associated with higher HCV prevalence regardless of duration of injecting, with the exception of IDUs who have 15 or more years injecting experience. In this group, recent risk behavior had no relationship to prevalence. In summary, our findings suggest a persistent HCV epidemic despite significant harm reduction efforts in Australia since the mid-1980s, with HIV incidence effectively constant in successive initiation cohorts.