Research has provided substantial evidences that heat shock proteins (HSPs) play essential roles in extreme physiological conditions. Heat shock transcription factors (HSFs) are important HSPs regulators, but their functions are poorly understood, particularly in Populus and Medicago. In this study, a comprehensive bioinformatics analysis of the HSFs was performed in Populus trichocarpa and Medicago truncatula. Twenty-eight Populus HSFs and 16 Medicago HSFs were identified, and comparative analyzes of the two plants were carried out subsequently. HSFs were divided into three different classes and they were diverse and complicated transcription factors. The results of semi-quantitative RT-PCR in Populus suggested six genes (PtHSF-03, PtHSF-13, PtHSF-15, PtHSF-21, PtHSF-22 and PtHSF-23) were markedly increased by heat stress. The results presented here provide an important clue for cloning, expression and functional studies of the HSFs in Populus and Medicago.