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 gene family has been systematically identified and analyzed in many plants but it is not in the tetraploid alfalfa genome. We detected 104 genes (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 s and tissue-wide expression for 28 s. Based on transcriptomic data analysis, 21, 11 and 27 s responded to drought stress, cold stress and salt stress, respectively, with seven responding to all three. According to RT-PCR, / expression gradually increased with cold, salt and drought stress condition duration; 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 s and for genetic improvement of stress resistance in alfalfa.