In recent years, immune repertoire profiling with high-throughput sequencing (HTS) has advanced our understanding of adaptive immunity. However, fast progress in the field applied mostly to human and mouse research, with only few studies devoted to other model vertebrates. We present the first in-depth characterization of the T-cell receptor (TCR) repertoire in a non-model mammal (bank vole, Myodes glareolus), widely used in ecological and evolutionary research. We used RNA from spleens, 5′RACE and HTS to describe V and J segments of TCRβ, qualitatively characterize preferential V–J segment usage and CDR3 length distribution. Overall orthology to murine genes was preserved, with 11 J and 37 V genes found in voles (although 3 V genes lacked a close orthologue). Further, we implemented unique molecular identifiers for quantitative analysis of CDR3 repertoire with stringent error correction. A conservative, lower bound estimation of the TCRβ repertoire was similar to that found for mice (1.7–2.3 × 10⁵ clonotypes). We hope that by providing an easy-to-follow molecular protocol and on-line bioinformatics tools that do not require reference sequences (AmpliTCR and AmpliCDR3), we will encourage HTS immune repertoire profiling in other non-model vertebrates, thus opening new research avenues in e.g. comparative immunology, ecology and evolutionary biology.