The most high-risk population for HCV transmission worldwide today are intravenous drug users. HCV genotypes in the general population in Cyprus demonstrate a polyphyletic infection and include subtypes associated with intravenous drug users. The prevalence of HCV, HBV, and HIV infection, HCV genotypes and risk factors among intravenous drug users in Cyprus were investigated here for the first time. Blood samples and interviews were obtained from 40 consenting users in treatment centers, and were tested for HCV, HBV, and HIV antibodies. On the HCV-positive samples, viral RNA extraction, RT-PCR and sequencing were performed. Phylogenetic analysis determined subtype and any relationships with database sequences and statistical analysis determined any correlation of risk factors with HCV infection. The prevalence of HCV infection was 50%, but no HBV or HIV infections were found. Of the PCR-positive samples, eight (57%) were genotype 3a, and six (43%) were 1b. No other subtypes, recombinant strains or mixed infections were observed. The phylogenetic analysis of the injecting drug users' strains against database sequences observed no clustering, which does not allow determination of transmission route, possibly due to a limitation of sequences in the database. However, three clusters were discovered among the drug users' sequences, revealing small groups who possibly share injecting equipment. Statistical analysis showed the risk factor associated with HCV infection is drug use duration. Overall, the polyphyletic nature of HCV infection in Cyprus is confirmed, but the transmission route remains unknown. These findings highlight the need for harm-reduction strategies to reduce HCV transmission. J. Med. Virol. 82:263-270, 2010. (C) 2009 Wiley-Liss, Inc.