Abiotic stresses extensively reduce agricultural crop production globally. For functional genomics studies, it is important to understand the dynamic expression profiles of transcribed genes in different tissues, stages of development and in response to environmental stimuli. Transcriptomics studies have contributed significantly to the understanding of the intricate dynamics of gene expression during stress conditions, unearthing novel stress-responsive candidate genes and signaling pathways. RNA sequencing (RNA-Seq) provides a far more precise measurement of levels of transcripts and their isoforms than other methods. Furthermore, comparative transcriptomics have provided more information about plants’ response to diverse stresses. Herein, we review studies that have used RNA-Seq profiling mainly in rice, barley and wheat to focus on their responses to the abiotic stresses including drought, salinity, heat, cold and heavy metals, which are important for developing tolerance mechanisms and designing climate-smart crops. Meanwhile, the utilization of pan-genomes in RNA-Seq profiling of rice and the available online RNA-Seq databases for the above-mentioned crops and their effective utilization are outlined. The acquired knowledge from RNA-Seq studies can support breeding for the identification of good agronomic traits in crop genome, to improve abiotic stress tolerance and crop yield.