Acquired thermotolerance in plants refers to the ability to cope with lethal high temperature following acclimatization at sublethal high temperatures. Acquired thermotolerance reflects an actual thermotolerance mechanism naturally occuring in plants and has been extensively used inthermotolerant line identification. In recent years, great progress has been achieved in the elucidation of biochemical, physiological, and molecular mechanisms of thermotolerance acquisition by using genomic approaches, including microarray analysis and mutation, knockout, and overexpression of related genes. Heat shock proteins (HSPs), such as Hsp 101 BOBBER 1, and Hsa32, have been shown to be important for inducement and maintenance of acquired thermotolerance. Downstream target genes and upstream regulation factors of HsfA2 including Hsa32, Apx2, small ubiquitin-like modifier proteins, FK506-binding proteins ROFI (FKBP62) and ROF2 (FKBP65), and heat shock transcription factor binding protein, have been revealed to be involved in thermotolerance acquisition regulation. Moreover, the role of abscisic acid, ethylene, ethylene, hydrogen peroxide, and salicylic acid in acquired thermotolerance has been demonstrated by molecular evidence from Arabidopsis mutants and transgenic lines. Most importantly, different molecular mechanisms of thermotolerance acquisition have benn shown to underlie various acclimatzation methods. Establishment of an experimental system similar to natural conditions is important for further explorationof natural thermotolerance mechanisms.