We report in this study the design and validation of a Pan-Vbeta primer that in combination with Cbeta-specific primers enables the amplification, in a single semi-nested PCR, of TCRbeta chains expressed by bovine T-cell clones irrespective of the expressed Vbeta sequence. Using the Pan-Vbeta primer we examined the TCRbeta chains expressed by 16 Theileria parva-specific clones that had not been previously analysed. TCRbeta chain sequence was obtained from 15 of the clones following direct sequencing of the PCR product, whilst the other clone appeared to express 2 different TCRbeta chains which were characterised following sub-cloning of the PCR product. We have also successfully used the Pan-Vbeta primer to amplify the TCRbeta chains expressed by 19 T-cell clones, on which previous analysis using Vbeta-subfamily-specific primers had failed to do. Sequencing of these TCRbeta chains has identified members of 2 novel bovine Vbeta subfamilies-Vbeta5 and VbetaX. This method offers a simple and rapid method of analyzing the TCRbeta chains of bovine T-cell clones that has many potential applications in the investigation of bovine T-cell responses.