Hepatitis C virus (HCV) genotype 1a and 3a partial NS5B gene segment sequences obtained from 154 HCV-infected injection drug users were studied to determine the extent to which HCV transmission occurs between injection drug user communities in London, Edinburgh, Glasgow (United Kingdom), Marseilles (France), and Melbourne. Phylogenetic relationships between sequences were analyzed by conventional methods and by a recently developed method that numerically scores the extent of sequence segregation between groups through calculation of association indices. The association indices revealed that none of the cities sampled support an HCV population that is completely isolated from that circulating in the other cities. Sequences from Melbourne were most isolated, whereas those from London were most dispersed. This suggests that HCV transmission between these cities occurs, with London playing a pivotal role. The degree of city-specific segregation of HCV subtype 1a sequences was linearly related to that of subtype 3a, indicating that these subtypes have spread through similar transmission networks.