Kentucky bluegrass (Poa pratensis) is strongly resistant to cold stress. Although the molecular mechanism of the plant response to cold stress has been widely documented in model plants, little is known about the cold tolerance of Kentucky bluegrass at the genomic level. Here, we compared the transcriptomes of Kentucky bluegrass under cold treatment (-5°C) and a control treatment (at 20°C) by RNA-seq and de novo assembly. Totally 75,934 unigenes were generated, among which 53,762 were successfully annotated in public databases. Upon comparing the transcriptomes of the control and cold-treated plants, 3,896 unigenes were identified as differentially expressed. Among these genes, 2,410 were down-regulated and 1,486 were up-regulated in the cold-treated plants. A few previously reported cold-induced proteins, antioxidant enzymes, and osmoregulation proteins were identified, and their expression levels were estimated. Moreover, ten differentially expressed genes were selected for qRT-PCR verification. Their expression patterns were consistent with the results of the RNA-seq. Additionally, the transcription factor families, i.e., ethylene response factors, heat stress transcription factors, NAC proteins, WRKY domaincontaining proteins, and auxin response factors, were identified as differentially expressed genes. The identification of these involved genes will facilitate studies on the transcriptional regulation of cold tolerance in plants.