Influenza B viruses fall in two antigenically distinct lineages (B/Victoria/2/1987 and B/Yamagata/16/1988 lineage) that co-circulate with influenza A viruses of the H3N2 and H1N1 subtype during seasonal epidemics. Infections with influenza B viruses contribute considerably to morbidity and mortality in the human population. Influenza B virus neutralizing antibodies, elicited by natural infections or vaccination, poorly cross-react with viruses of the opposing influenza B lineage. Therefore, there is an increased interest in identifying other correlates of protection which could aid the development of broadly-protective vaccines. BLAST analysis revealed high sequence identity of all viral proteins. With two online epitope prediction algorithms, putative conserved epitopes relevant for study subjects used in the present study, were predicted. The cross-reactivity of influenza B virus-specific polyclonal CD8+ T lymphocyte populations, obtained from HLA-typed healthy study subjects, with intra-lineage drift variants and viruses of the opposing lineage was determined by assessing their in vitro interferon gamma (IFN-γ) response and lytic activity. Here, we show for the first time, that CD8+ T lymphocytes directed to viruses of the B/Victoria lineage cross-react with viruses of the B/Yamagata lineage and vice versa.