Hepatitis C virus (HCV) is a highly variable pathogen that frequently establisheschronic infection. This genetic variability is affected by the adaptive immune response but thecontribution of other host factors is unclear. Here, we examined the role played by interferonlambda-4 (IFN-l4) on HCV diversity; IFN-l4 plays a crucial role in spontaneous clearance orestablishment of chronicity following acute infection. We performed viral genome-wide associationstudies using human and viral data from 485 patients of white ancestry infected with HCVgenotype 3a. We demonstrate that combinations of host genetic variants, which determine IFN-l4protein production and activity, influence amino acid variation across the viral polyprotein - notrestricted to specific viral proteins or HLA restricted epitopes - and modulate viral load. We alsoobserved an association with viral di-nucleotide proportions. These results support a direct role forIFN-l4 in exerting selective pressure across the viral genome, possibly by a novel mechanism.