Wheat (Triticum aestivum L.) growing areas in many regions of the world are subject to heat waves which are predicted to increase in frequency because of climate change. The engineering of crop plants can be a useful strategy to mitigate heat stress-caused yield losses. Previously, we have shown that heat shock factor subclass C (TaHsfC2a-B)-overexpression significantly increased the survival of heat-stressed wheat seedlings. Although previous studies have shown that the overexpression of Hsf genes enhanced the survival of plants under heat stress, the molecular mechanisms are largely unknown. To understand the underlying molecular mechanisms involved in this response, a comparative analysis of the root transcriptomes of untransformed control and TaHsfC2a-overexpressing wheat lines by RNA-sequencing have been performed. The results of RNA-sequencing indicated that the roots of TaHsfC2a-overexpressing wheat seedlings showed lower transcripts of hydrogen peroxide-producing peroxidases, which corresponds to the reduced accumulation of hydrogen peroxide along the roots. In addition, suites of genes from iron transport and nicotianamine-related gene ontology categories showed lower transcript abundance in the roots of TaHsfC2a-overexpressing wheat roots than in the untransformed control line following heat stress, which are in accordance with the reduction in iron accumulation in the roots of transgenic plants under heat stress. Overall, these results suggested the existence of ferroptosis-like cell death under heat stress in wheat roots, and that TaHsfC2a is a key player in this mechanism. To date, this is the first evidence to show that a Hsf gene plays a key role in ferroptosis under heat stress in plants. In future, the role of Hsf genes could be further studied on ferroptosis in plants to identify root-based marker genes to screen for heat-tolerant genotypes.