Temperature is one of the most important abiotic factors defining the yield potential of temperate cereal crops such as barley. The regulators of heat shock response (HSR); Heat shock factors (HSFs) modulate the transcription level of heat responsive genes in order to protect the plants against heat stress. In the present study, a heat shock factor from wheat (TaHSFA6b) is overexpressed in barley for providing thermotolerance. Transgenic barley lines overexpressing TaHSFA6b showed significant improvement in thermotolerance. The constitutive overexpression of TaHSFA6b gene upregulated the expression of major heat shock protein genes as well as other abiotic stress responsive genes. RNA-seq and qRT-PCR analysis showed upregulation of HSPs, chaperonins, DNAJ, LEA proteins and genes related to anti-oxidative enzymes in transgenic lines. Excessive generation and accumulation of ROS occurred in wild type plants during heat stress; however, the transgenic lines reflected improved ROS homeostasis mechanisms in the form of significantly low ROS accumulation under high temperature. There were no negative phenotypic changes in overexpression lines. The present study suggests that TaHSFA6b is one of the major regulators of HSR as it showed the capacity to alter the expression patterns of main defense related genes and enhance the thermotolerance of this cereal crops.