Hepatitis C virus (HCV) infects 2–3% of the world population and intravenous drug consumption is the leading cause of transmission in industrialized countries. The unavailability of data on the molecular epidemiology of HCV infection in Portugal prompted the study of HCV subtypes circulating among intravenous drug users residing in the Lisbon metropolitan area and sampled about 10 years apart (1998–2000 and 2008–2009). Partial coding sequences for E1 and/or NS5B were obtained from 124 individuals with HCV viremia, both mono-infected and co-infected with HIV. Phylogenetic analysis showed that, for both time periods, the most prevalent subtypes were 1a and 3a, found, altogether, in 64.9% and 71.6% of the individuals, respectively for the first and the second sampling periods. However, genotype 4 viruses (subtypes 4a and 4d), introduced later, as inferred by comparison of intra-subtype genetic distances, were also relatively frequent even one decade ago (24.6%). This HCV subtype profile for Portuguese intravenous drug users is in agreement with those described for other southern European countries when in association with drug consumption. With the exception of subtype 1b, phylogenetic trees did not show clustering of the Portuguese sequences, but rather phylogenetic mixing of HCV sequences from different geographic origins, as described previously in other Western countries and suggestive of a large international transmission network. Consistent with the low recombination rates reported for HCV, only one sample revealed discordant subtypes for the two regions analyzed (4d in E1 and 4a in NS5B), representing a potential new recombinant that deserves further analysis. J. Med. Virol. 83:608–615, 2011. © 2011 Wiley-Liss, Inc.