Plants have evolved efficient mechanisms for adapting to temperature fluctuations, known as heat stress response and heat stress memory. Although the transcriptional regulatory network of plant heat stress response has been established, little is known about the genome-wide transcriptional changes oc-curring within the first several minutes after heat shock. Here, we investigated the nascent RNA and mature messenger RNA (mRNA) from plant leaf tis-sues exposed to 5 min of heat shock treatment using global run-on sequencing and RNA sequencing methods. Only a small group of genes were up- or downregulated at both the nascent RNA and mRNA levels. Primed plants that were already exposed to mild heat stress exhibited a more drastic alteration at multiple transcriptional steps than na?ve plants that had not experienced heat stress. Upon heat shock, we also observed the following: (i) engaged RNA polymerase II accumulated downstream of tran-scription start sites;(ii) 5′pausing release was a rate-limiting step for the induction of some heat shock protein genes; (iii) numerous genes switched tran-scription modes;(iv) pervasive read-through was in-duced at terminators; and (v) heat stress memory occurs at multiple steps of the transcription cycle, such as at Pol II recruitment, 5′pausing, elongation, and termination.