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Hepatitis C virus (HCV) is a highly diverse pathogen that is classified into seven distinct genotypes. Simultaneous or sequential reinfection with multiple HCV genotypes is recognized in high-risk populations, such as injecting drug users (IDUs). Multiple infection is of clinical concern as different genotypes have various sensitivities to current antiviral therapies. Therefore, a better understanding of the frequency of multiple infection and of the genotypes currently being transmitted is clinically relevant. An Australian cohort of IDUs (n = 123), identified with primary incident infection, was followed for multiple infection by regular HCV RNA testing between 2005 and 2013. A total of 354 samples were tested. Sequencing of primary incident infections revealed that genotype 3a was the most common circulating genotype, followed by genotype 1a. Examination of longitudinally collected samples identified complex patterns of multiple infection, including reinfection and superinfection. In those with multiple infection, there was no apparent evidence of homotypic immunity conferring protection against reinfection of the same subtype. This study revealed frequent multiple infection in a high-risk prisoner cohort, illustrating the complex nature of HCV infection and reinfection and highlighting the need for pan-genotypic antiviral therapies., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)