Background Initial data on the molecular epidemiology of HCV infection in Cyprus showed a highly polyphyletic infection and multiple points of introduction into the general population. The continuation and expansion of this investigation is presented here including high risk groups. Findings The samples include additional subjects from the general population, a group of inmates and HIV/HCV coinfected individuals, whose strains were amplified by RT-PCR and sequenced in partial Core-E1 and NS5B regions. The results confirm the broad genotype distribution and polyphyletic infection on the island, and no new subtypes were found. Monophyletic clusters between strains of the prisoners and the injecting drug users imply sharing of infected equipment, and highlight the risk of widespread transmission in these cohorts, although no spill-over to the general population was observed. Conclusions The results of this study underline the impact of population movements and high-risk population groups on the changing molecular epidemiology of HCV, with strains moving to Europe from Asia, Africa and Eastern Europe by means of immigration and modern transmission routes.