Background: Injecting drug users (IDU) have a key role in Hepatitis C Virus (HCV) epidemiology. Young/short-term IDUs constitute a target group for preventive/harm reduction interventions. Objectives: To investigate HCV transmission among young/short-term (ST) and long-term (LT) IDUs, from the perspective of epidemiology and molecular biology. Study design: Cross-sectional study assessing the prevalence of HCV infection/genotypes, as well as risk behaviours/practices among IDUs from Rio de Janeiro. Phylogenetic analyses were performed and the extent of segregation between sequences was quantified by the Association Index. Results: ST were more likely to engage into needle-sharing (p = .021) and LT to attend Needle Exchange Programs (p = .006). HCV prevalence was 10.1% vs. 23.4% among initiates and LT, respectively (p < .001). Older age vs. imprisonment and longer duration of IDU career were independent predictors for HCV infection among ST and LT, respectively. Among the latter, NEP attendance was inversely associated with viral infection. HCV3a infectionswere the most prevalent. A moderate extent of phylogenetic segregation between sequences was found, suggestive of transmission between IDU subgroups. Conclusions: The lower HCV prevalence among young/short-term IDUs cannot be viewed with complacency, due to their frequent engagement into direct/indirect sharing practices and the ongoing transmission between IDU subsets. To avert new infections, preventive/harm reduction policies must be tailored to empirical findings.