Tall fescue is one of the primary source of forage for livestock. It grows well in the marginal soils of the temperate zones. It hosts a fungal endophyte (Epichloë coenophiala), which helps the plants to tolerate abiotic and biotic stresses. The genetics and biology underlying mechanism of freezing stress tolerance of tall fescue is still unknown, due to its complex genetic background and outbreeding modes of pollination, limited genomic, and transcriptomic resources. The aim of this study was to identify differentially expressed genes (DEGs) in two tissues between novel endophyte-positive (E+) and endophyte-free (E-) tall fescue genotypes at three diurnal time points; in the morning (-3.0 to 0.5°C), afternoon (11 to 12°C), and evening (12 − 10°C) in the field environment, by exploring the transcriptional landscape via RNA sequencing. For the first time, we generated 226,054 and 224,376 transcripts from E + and E- Texoma MaxQ II tall fescue, respectively by de novo assembly. The upregulated transcripts were detected fewer than the downregulated ones in both tissues (S: 803 up and 878 down; L: 783 up and 846 down) under the freezing temperatures in the morning. By Gene Ontology enrichment analysis, 10 GO terms were found only under the freezing stress in the morning. Metabolic pathway and biosynthesis of secondary metabolites genes showed lowest number of DEGs under morning freezing stress and highest number in evening cold condition by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis. The DEGs expressed under morning stress condition and the nine candidate genes that we identified using GO analysis, might be the possible route underlying cold tolerance in tall fescue.