Antiviral CD8(+) T cells are a key component of the adaptive immune system against hepatitis C virus (HCV). For the development of immune therapies, it is essential to understand how CD8(+) T cells contribute to clearance of infection and why they fail so often. A mechanism for secondary failure is mutational escape of the virus. However, some substitutions in viral epitopes are associated with fitness costs and often require compensatory mutations. We hypothesized that compensatory mutations may point toward epitopes under particularly strong selection pressure that may be beneficial for vaccine design because of a higher genetic barrier to escape. We previously identified two HLA-B*15-restricted CD8(+) epitopes in NS5B (LLRHHNMVY(2450-2458) and SQRQKKVTF(2466-2474)), based on sequence analysis of a large HCV genotype 1b outbreak. Both epitopes are targeted in about 70% of HLA-B*15-positive individuals exposed to HCV. Reproducible selection of escape mutations was confirmed in an independent multicenter cohort in the present study. Interestingly, mutations were also selected in the epitope flanking region, suggesting that compensatory evolution may play a role. Covariation analysis of sequences from the database confirmed a significant association between escape mutations inside one of the epitopes (H2454R and M2456L) and substitutions in the epitope flanking region (S2439T and K2440Q). Functional analysis with the subgenomic replicon Con1 confirmed that the primary escape mutations impaired viral replication, while fitness was restored by the additional substitutions in the epitope flanking region. We concluded that selection of escape mutations inside an HLA-B*15 epitope requires secondary substitutions in the epitope flanking region that compensate for fitness costs.