The Influenza B virus (IBV) can pose a significant threat to global health. Despite this, IBV is understudied compared to Influenza A virus (IAV). CD8+ T cells have proven highly effective in reducing influenza disease severity. In addition, pre-existing immune responses towards IAV epitopes may provide protection against homologous IBV-derived peptides due to T cell cross-reactivity. To exploit the advantages of T cells for future vaccine developments, a better understanding of the IBV-derived peptide presentation by the highly polymorphic Human Leukocyte Antigen (HLA) is required. We previously determined that the IAV-derived PB1177-A peptide was presented by the HLA-B18:01 molecule and induced CD8+ T cell responses. Here we assessed the PB1177-A IBV homologue (PB1177-B). Intracellular cytokine staining assays showed that PB1177-B was unable to activate CD8+ T cells from several HLA-B18:01+ samples tested. We determined that the IAV- and IBV-derived PB1177 adopted different stability and conformation in the cleft of HLA-B18:01. Molecular dynamics analysis on the crystal structure showed that PB1177-B had a central flexible region with a large hydrophobic patch formed by two phenylalanine residues, not present in PB1177-A. The flexibility and the lower stability of the HLA-B18:01-PB1177-B complex may hinder CD8+ T cell receptor binding, underpinning the lack of CD8+ T cell activation observed. This provides additional insights into the differences between IAV- and IBV-specific CD8+ T cell responses.