TCRγδ T cells are a major lymphocyte population in chickens, but their response to ALV-J and recognized antigens remains poorly understood. Here, we report an increase in the proportion and activation state of TCRγδ⁺CD8α⁺ T cells in vivo and in vitro cultured T cells from 2 chicken lines (MHC homozygous H-B21 lines and outbred G-WL line), with the earlier and stronger proliferative response observed in the H-B21 chickens. Activated TCRγδ⁺CD8α⁺ T cells exhibited ‘cytolytic’ potential, as detected by transcription levels of cytotoxic genes using quantitative reverse transcription PCR (qRT-PCR), and IFN-γ protein levels using ELISPOT and intracellular cytokine staining (ICS) assays, with a more significant response in the H-B21 chickens. Furthermore, the viral structural proteins Gag and Pol were identified as major target proteins recognized by chicken TCRγδ⁺CD8α⁺ T cells. Further ELISPOT and ICS analyses mapped the key peptides to Gag_187–215 and Pol_417–445 recognized by chicken TCRγδ⁺CD8α⁺ T cells against ALV-J infection. Collectively, these findings confirm for the first time that TCRγδ⁺CD8α⁺ T cells can recognize viral antigens and reveal the dominant peptides on ALV-J, enhancing our understanding of anti-ALV-J T cell responses and advancing vaccine design strategies against ALV-J.