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Heat shock transcription factors (HSFs) activate heat shock protein gene expression by binding their promoters in response to heat stress and are considered to be pivotal transcription factors in plants. <i>Eucalyptus</i> is a superior source of fuel and commercial wood. During its growth, high temperature or other abiotic stresses could impact its defense capability and growth. <i>Hsf</i> genes have been cloned and sequenced in many plants, but rarely in <i>Eucalyptus</i>. In this study, we used bioinformatics methods to analyze and identify <i>Eucalyptus</i> <i>Hsf</i> genes, their chromosomal localization and structure. The phylogenetic relationship and conserved domains of their encoded proteins were further analyzed. A total of 36 <i>Hsf</i> genes were identified and authenticated from <i>Eucalyptus</i>, which were scattered across 11 chromosomes. They could be classified into three classes (A, B and C). Additionally, a large number of stress-related cis-regulatory elements were identified in the upstream promoter sequence of <i>HSF</i>, and cis-acting element analysis indicated that the expression of <i>EgHsf</i> may be regulated by plant growth and development, metabolism, hormones and stress responses. The expression profiles of five representative <i>Hsf</i> genes, <i>EgHsf4</i>, <i>EgHsf9</i>, <i>EgHsf13</i>, <i>EgHsf24</i> and <i>EgHsf32</i>, under salt and temperature stresses were examined by qRT-PCR. The results show that the expression pattern of class B genes (<i>EgHsf4</i>, <i>EgHsf24</i> and <i>EgHsf32</i>) was more tolerant to abiotic stresses than that of class A genes (<i>EgHsf9</i> and <i>EgHsf13</i>). However, the expressions of all tested <i>Hsf</i> genes in six tissues were at different levels. Finally, we investigated the network of interplay between genes, and the results suggest that there may be synergistic effects between different <i>Hsf</i> genes in response to abiotic stresses. We conclude that the <i>Hsf</i> gene family played an important role in the growth and developmental processes of <i>Eucalyptus</i> and could be vital for maintaining cell homeostasis against external stresses. This study provides basic information on the members of the <i>Hsf</i> gene family in <i>Eucalyptus</i> and lays the foundation for the functional identification of related genes and the further investigation of their biological functions in plant stress regulation.