AbstractHepatitis C virus (HCV)-specific T cell responses are critical for immune control of infection. Viral adaptation to these responses, via mutations within regions of the virus targeted by CD8⁺ T cells, is associated with viral persistence. However, identifying viral adaptation to HCV-specific CD4⁺ T cell responses has been difficult although key to understanding anti-HCV immunity. In this context, HCV sequence and host genotype from a single source HCV genotype 1B cohort (n = 63) were analyzed to identify viral changes associated with specific human leucocyte antigen (HLA) class II alleles, as these variable host molecules determine the set of viral peptides presented to CD4⁺ T cells. Eight sites across the HCV genome were associated with HLA class II alleles implicated in infection outcome in this cohort (p ≤ 0.01; Fisher’s exact test). We extended this analysis to chronic HCV infection (n = 351) for the common genotypes 1A and 3A. Variation at 38 sites across the HCV genome were associated with specific HLA class II alleles with no overlap between genotypes, suggestive of genotype-specific T cell targets, which has important implications for vaccine design. Here we show evidence of HCV adaptation to HLA class II-restricted CD4⁺ T cell pressure across the HCV genome in chronic HCV infection without a priori knowledge of CD4⁺ T cell epitopes.