It has become clear in recent years that many genes in a given species may not be found in a single genotype thus using sequences from a single genotype as reference may not be adequate for various applications. In this study we constructed a pan-transcriptome for barley by de novo assembling 288 sets of RNA-seq data from 32 cultivated barley genotypes and 31 wild barley genotypes. The pan-transcriptome consists of 756,632 transcripts with an average N50 length of 1240 bp. Of these, 289,697 (38.2%) were not found in the genome of the international reference genotype Morex. The novel transcripts are enriched with genes associated with responses to different stresses and stimuli. At the pan-transcriptome level, genotypes of wild barley have a higher proportion of disease resistance genes than cultivated ones. We demonstrate that the use of the pan-transcriptome dramatically improved the efficiency in detecting variation in barley. Analysing the pan-transcriptome also found that, compared with those in other categories, disease resistance genes have gone through stronger selective pressures during domestication.