The 5' untranslated region (5'UTR) of hepatitis C virus (HCV) is structured into four domains (I-IV) with numerous genotype-specific nucleotides. It is unknown whether the polymorphisms confer genotype-specific functions to the 5'UTR. Using viable JFH1-based Core-NS2 recombinants, we developed and characterized HCV genotypes 1-7 recombinants with highly diverse 5'UTRs (genotypes 1a and 3a), 2a recombinants (J6/JFH1) with 5'UTR of genotypes 1-6 or with heterotypic chimeric (1a/3a and 3a/1a) 5'UTR domains I, II or III, and 1a recombinants with 5'UTR domain I of genotypes 1-6. All were fully functional in Huh7.5 cells; therefore, the 5'UTR apparently functions in a non-genotype-specific manner in HCV production in vitro. However, adenine at the 5'-terminus was required. We demonstrated that J6/JFH1 with 5'UTR of genotypes 1-6 responded similarly to interferon-a2b. This study provides novel insight into the role of the 5'UTR in the HCV life cycle and facilitates HCV basic research and testing of 5'UTR-targeting antivirals.