RNA-Seq using second-generation sequencing technologies permits generation of a reference unigene set for a given species, in the absence of a well-annotated genome sequence, supporting functional genomics studies, gene characterisation and detailed expression analysis for specific morphophysiological or environmental stress response traits. A reference unigene set for lentil has been developed, consisting of 58,986 contigs and scaffolds with an N50 length of 1719 bp. Comparison to gene complements from related species, reference protein databases, previously published lentil transcriptomes and a draft genome sequence validated the current dataset in terms of degree of completeness and utility. A large proportion (98%) of unigenes were expressed in more than one tissue, at varying levels. Candidate genes associated with mechanisms of tolerance to both boron toxicity and time of flowering were identified, which can eventually be used for the development of gene-based markers. This study has provided a comprehensive, assembled and annotated reference gene set for lentil that can be used for multiple applications, permitting identification of genes for pathway-specific expression analysis, genetic modification approaches, development of resources for genotypic analysis, and assistance in the annotation of a future lentil genome sequence.