Perennial ryegrass (PR; Lolium perenne L.) and meadow fescue (MF; Festuca pratensis Huds.) are important grass species for forage production around the world. These species are genetically related but show different responses to biotic and abiotic stresses such as freezing: PR is severely damaged by cold stress, but MF is not. Recently, next-generation sequencing technology (e.g. 454 sequencing) has been used to profile the genome-wide transcriptomics of various plant species. To clarify the genetic differences behind the different cold responses of MF and PR, we developed an effective method for comparing transcriptome data during cold acclimation. The expressed sequence tags (ESTs) from cold-acclimated MF and PR were obtained by means of minimum-scale 454 sequencing runs. After trimming based on quality scores, these redundant ESTs were assembled into a total of 1527 contigs and singletons for MF and 1060 for PR. Subsequently, we clustered the contigs and singletons into 1082 (MF) and 661 (PR) clusters based on sequence similarity, and we used the EST numbers for the corresponding clusters to compare levels of gene expression. We observed four clusters (wheat germ agglutinin isolectin, γ-thionin, jasmonate-induced protein homolog and germin) with markedly different numbers of ESTs between MF and PR. The species-specific expression of genes corresponding to these four clusters was confirmed by means of reverse-transcriptase polymerase chain reaction analysis.