T-cell receptor (TCR) usage has an important role in determining the outcome of CD8+ cytotoxic T-lymphocyte responses to viruses and other pathogens. However, the characterization of TCR usage from which such conclusions are drawn is based on exclusive analysis of either the TCRalpha chain or, more commonly, the TCRbeta chain. Here, we have used a multiplexed reverse transcription-PCR protocol to analyse the CDR3 regions of both TCRalpha and beta chains from single naive or immune epitope-specific cells to provide a comprehensive picture of epitope-specific TCR usage and selection into the immune response. Analysis of TCR repertoires specific for three influenza-derived epitopes (DbNP366, DbPA224 and DbPB1-F262) showed preferential usage of particular TCRalphabeta proteins in the immune repertoire relative to the naive repertoire, in some cases, resulting in a complete shift in TRBV preference or CDR3 length, and restricted repertoire diversity. The NP366-specific TCRalphabeta repertoire, previously defined as clonally restricted based on TCRbeta analysis, was similarly diverse as the PA224- and PB1-F262-specific repertoires. Intriguingly, preferred TCR characteristics (variable gene usage, CDR3 length and junctional gene usage) appeared to be able to confer specificity either independently or in concert with one another, depending on the epitope specificity. These data have implications for established correlations between the nature of the TCR repertoire and response outcomes after infection, and suggest that analysis of a subset of cells or a single TCR chain does not accurately depict the nature of the antigen-specific TCRalphabeta repertoire.Immunology and Cell Biology advance online publication, 21 April 2015; doi:10.1038/icb.2015.44.