Heat shock transcription factors (HSFs) contribute significantly to thermotolerance acclimation. Here, we identified and cloned a putative HSF gene (HSFA2h) of 1218 nucleotide (acc. no. KP257297.1) from wheat cv. HD2985 using a de novo transcriptomic approach and predicted sHSP as its potential target. The expression of HSFA2h and its target gene (HSP17) was observed at the maximum level in leaf tissue under heat stress (HS), as compared to the control. The HSFA2h-pRI101 binary construct was mobilized in Arabidopsis, and further screening of T3 transgenic lines showed improved tolerance at an HS of 38 °C compared with wild type (WT). The expression of HSFA2h was observed to be 2.9- to 3.7-fold higher in different Arabidopsis transgenic lines under HS. HSFA2h and its target gene transcripts (HSP18.2 in the case of Arabidopsis) were observed to be abundant in transgenic Arabidopsis plants under HS. We observed a positive correlation between the expression of HSFA2h and HSP18.2 under HS. Evaluation of transgenic lines using different physio-biochemical traits linked with thermotolerance showed better performance of HS-treated transgenic Arabidopsis plants compared with WT. There is a need to further characterize the gene regulatory network (GRN) of HSFA2h and sHSP in order to modulate the HS tolerance of wheat and other agriculturally important crops.