VDJ rearrangement at the teleost TCRbeta locus leads to a highly diverse repertoire of junctions for each VbetaJbeta combination. From a rainbow trout 5' RACE library of TCRbeta transcripts, 47 clones encompassing a full Vbeta-Dbeta-Jbeta-Cbeta sequence were selected and analyzed. A similarity analysis of the sequences evidenced 10 Vbeta families, of which 6 were not previously described. Immunoscope and sequence analysis of the Vbeta-Dbeta-Jbeta junctions of the new families confirmed that they create a polyclonal and diverse repertoire. Multiple alignments showed that rainbow trout Vbetas possess most of the conserved residues typical of Vbeta segments. However, this study revealed a high complementarity-determining region 2 (CDR2) and CDR1 length diversity among rainbow trout Vbeta families, suggesting that the spatial orientation of the TCR could fluctuate in the TCR/peptide/MHC complex, depending on the Vbeta expressed. Among the new Vbeta families, Vbeta6 displayed the strongest deviance from typical hypervariable CDR1 and CDR2 loops, with an unusually short CDR2. Moreover, the Vbeta6 sequence is overall divergent from typical Vbeta sequence, raising the question of its functional relevance. Immunoscope experiments identified a Vbeta6-Jbeta3 junction, which was amplified during the response against viral hemorrhagic septicemia virus, a fish rhabdovirus. Vbeta6 seems therefore to be expressed functionally in a selected TCR. However, the shorter Vbeta6 transcripts produced through an alternative splicing lack the C', C", D, and E strands of the Vbeta domain and are probably nonfunctional.