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Heat shock transcription factors (HSFs) are important regulatory factors in plant stress responses to various biotic and abiotic stresses and play important roles in growth and development. The <i>HSF</i> gene family has been systematically identified and analyzed in many plants but it is not in the tetraploid alfalfa genome. We detected 104 <i>HSF</i> genes (<i>MsHSF</i>s) in the tetraploid alfalfa genome (“Xinjiangdaye” reference genome) and classified them into three subgroups: 68 in HSFA, 35 in HSFB and 1 in HSFC subgroups. Basic bioinformatics analysis, including genome location, protein sequence length, protein molecular weight and conserved motif identification, was conducted. Gene expression analysis revealed tissue-specific expression for 13 <i>MsHSF</i>s and tissue-wide expression for 28 <i>MsHSF</i>s. Based on transcriptomic data analysis, 21, 11 and 27 <i>MsHSF</i>s responded to drought stress, cold stress and salt stress, respectively, with seven responding to all three. According to RT–PCR, <i>MsHSF27</i>/<i>33</i> expression gradually increased with cold, salt and drought stress condition duration; <i>MsHSF6</i> expression increased over time under salt and drought stress conditions but decreased under cold stress. Our results provide key information for further functional analysis of <i>MsHSF</i>s and for genetic improvement of stress resistance in alfalfa.