AbstractWe report the first mountain hare (Lepus timidus) transcriptome, produced by de novo assembly of RNA-sequencing reads. Data were obtained from eight specimens sampled in two localities, Alps and Ireland. The mountain hare tends to be replaced by the invading European hare (Lepus europaeus) in their numerous contact zones where the species hybridize, which affects their gene pool to a yet unquantified degree. We characterize and annotate the mountain hare transcriptome, detect polymorphism in the two analysed populations and use previously published data on the European hare (three specimens, representing the European lineage of the species) to identify 4 672 putative diagnostic sites between the species. A subset of 85 random independent SNPs was successfully validated using PCR and Sanger sequencing. These valuable genomic resources can be used to design tools to assess population status and monitor hybridization between species.