This dataset contains the raw and processed data of the transcriptome of 12 grass species of the Panicoideae and Chloridoideae subfamilies under ambient conditions (32°C (day) and 18°C (night) and midday light intensity averaged 800 µmol m-2 s-1 during the growth period). Raw data was generated using Illumina Hi-Seq 2500 machine with 150 base pair paired end sequencing. Raw reads were then processed using Trimmomatic and Blue to both trim the adapters and correct any sequencing errors. Reads were assembled for each species using de novo assembly using Trinity. If there was a published genome, genome guided assembly was also used using Trinity, then both the genome guided and de novo assembly were combined using PASA to remove any redundancy. The data was generated from leaf discs collected from Western Sydney University and Australian National University glasshouses from 12 species; Leptochloa dubia, Leptochloa fusca, Astrebla pectinate, Chloris gayana, Panicum coloratum, Panicum antidotale, Panicum bisulcatum, Panicum monticola, Panicum virgatum, Axonopus fissifolius, Sorghum bicolor and Echinochloa frumentaceae. Samples were snap frozen in liquid nitrogen then RNA was extracted from frozen leaf discs using PureZol (BioRad). Aliquots (4 µg) of RNA were treated with DNase I (AMBION) following the manufacturer’s protocols. The RNA was purified by phenol-chloroform extraction and precipitation in ethanol and sodium acetate. Purified RNA was centrifuged and washed 3 times in 75% ethanol and resuspended in 50 µL of nuclease free H2O. Prior to library preparation, ribosomal RNA was removed using the RiboZero Plant rRNA kit (Epicentre). RNA-Seq was carried out on the extracted RNA at the NGS facility at Western Sydney University using an Illumina Hi-Seq 2500 machine with 150 base pair paired end sequencing. Between 30 and 40 million reads were returned per sample.